Biological samples
The study was performed with plasma samples obtained from pregnant women differentially exposed to P.f and/or T.g parasites in order to evaluate the joint antibody response to both pathogens. For this purpose, 150 plasmas, all preserved at −20 °C until use and originating from three different field studies, were selected (Table 3).
PlasDCty samples (Benin)
The PlasDCty (PDC) study took place from June to November 2021 in the maternity of Akassato. This locality in southern Benin (6° 30’ 0” North, 2° 22’ 0” East) is located in the Atlantic Department, 20 km northwest of Cotonou, the economic capital of Benin. The climate in this area is sub-equatorial, with two rainy seasons extending from April to July and October to November. Malaria is perennial, with transmission peaks during the rainy seasons. Anopheles gambiae and An. funestus are the main malaria vectors, and Plasmodium falciparumis the major infecting species69. The study was therefore conducted over the two rainy seasons. It was designed to evaluate the immune modulation of neonatal dendritic cells (DC) due to maternal infection and their ability to activate T cells to design a potential vaccine candidate able to fully activate the DC. Women were screened at delivery (n = 337) for malaria using PfHRP-II and Plasmodium LDH Rapid Diagnosis Test (RDT) to select all 29 pregnant women with confirmed P.f infection, based on microscopic and/or PCR results. Regarding microscopic examination, parasites and peripheral blood mononuclear cells were counted on 10% Giemsa-stained thick blood smears up to 500 leucocytes. If no parasite was detected, an extra 500 leucocytes were counted to ensure the negativity. Parasitemia was estimated using 8000 leucocytes per microliter of blood following the formula: parasites/µL blood = (No. of parasites counted x 8000 white blood cells/µL) / No. of white blood cells counted. For PCR, DNA was extracted from 200 µL of whole blood using DNeasy Blood kit (Qiagen, France) and used to detect Plasmodium species (P. falciparum, P. malariae and P. ovale) by nested PCR amplification of the 18s small subunit ribosomal RNA gene as previously published70. Inclusion criteria were women of 18 years old or older, a priori termed delivery with expected non-complication. Diagnostic testing with Platelia™ Toxo IgG (Bio-Rad, France) and Malaria EIA (BioRad, France) kits was performed to determine each woman’s toxoplasmosis and malaria serological status.
CoaLa samples (Benin)
The CoaLa (COA) study was carried out from February to early April 2018 at the maternity ward of the CHU-MEL (Centre Hospitalier et Universitaire de la Mère et de l’Enfant Lagune), located in the center of Cotonou, in the Littoral Department of Benin. Most women admitted to this hospital, specialized in gynecology, obstetrics and pediatrics, reside in Cotonou or the surrounding area. The climate is similar to what was described above, so the study occurred during the dry season, corresponding to low or no malaria pressure. It was designed as part of the implementation of a new diagnostic approach for congenital toxoplasmosis and concerned 106 women recruited at their last antenatal care visit, in their third trimester of pregnancy71. Inclusion criteria were: at least 18 years of age and pregnancy 7 months, clinical uncomplicated delivery planned at the maternity, and residing in neighborhoods close to CHU-MEL. A total of 95 plasma samples were available for the present study. Toxoplasmosis serology was performed with recomWell Toxoplasma IgG kits (Mikrogen Diagnostik, Germany) and verified by VIDAS® TOXO IgG II (bioMérieux, France) and malaria serological status was determined with Malaria EIA (Bio-Rad, France) kits.
TOXODIAG samples (France)
The TOXODIAG (TXD) study was carried out between 2018 and 2023 among 60 women performing prenatal follow-up and giving birth in the maternity wards of 5 hospitals from the AP-HP (Assistance Publique-Hôpitaux de Paris, France). Inclusion criteria were at least age 18 years and clinically uncomplicated delivery. The study aimed to identify women diagnosed with toxoplasma seroconversion during pregnancy as part of the implementation of a new diagnostic approach for congenital toxoplasmosis. Among the available collected samples during the study, 26 plasma samples corresponding to malaria non-exposed women were selected and divided into two groups according to the serological toxoplasmosis results delivered by the hospital biology laboratories. The malaria serological status was performed with Malaria EIA (Bio-Rad, France) kits in order to check that the women were seronegative for malaria.
Sequence analysis and structure prediction with bioinformatic tools
Sequence homology analysis
A Basic Local Alignment Search Tool (BLAST) was performed to identify homologies in public databases. This was followed by a global alignment using the MUSCLE program in MEGA X software (https://www.megasoftware.net/) to determine sequence similarities between P.f and T.g proteins targeting PfAMA1 and TgAMA1. The Vector Alignment Search Tool (VAST+) was used to identify structural similarities between P.f and T.g proteins.
Epitope prediction tools
An epitope prediction mapping was implemented by using the Immune Epitope Database (IEDB) Analysis Resource to understand antigen-antibody interactions. The database provides a comprehensive suite of bioinformatics tools for B-cell and T-cell epitope prediction. Conformational/discontinuous B-cell epitopes resulting from 3D protein structures (Protein Data Bank) were explored with the IEDB Disco Tope tool (Version 1.1) associated with CB Tope and BCPREDS servers for a robust selection of recurrent predicted epitopes. The default parameter was selected for the threshold. Structural analyses were handled by the PyMol software 3.0. Common B-cell epitopes were identified on the aligned PfAMA1 et TgAMA1 protein sequences.
Design of Recombinant antigens and expression vectors
The analysis of the protein sequences and the secondary structure prediction was established using ExPASy tools resources and particularly the TMHMM to predict transmembrane domains and signal peptides. The designed pET28a + expression vector, including selecting suitable restriction sites, was verified with Serial Cloner (Version 2.6.1).
Production of PfAMA1, TgAMA1, TgSAG1 and TgGRA7 recombinant antigens
Transformation and expression of the recombinant proteins in E. coli
The PfAMA1 sequence (Q7KQK5) was selected from the 3D7 strain in the PlasmoDB database. The 3D7 strain is a clone from the NF54 isolate, which originates from African regions72. 3D7 is a reference laboratory strain for in vitro drug testing and inhibition assays73. The genetic sequences of the selected recombinant antigens TgAMA1 (B6KAM0), TgSAG1 (A0A125YP09) and TgGRA7 (A0A125YI90) were identified in the ToxoDB database, referencing the type II Me49 strain. Type II strain was prioritized because of its worldwide dissemination notably in Europe and Africa and also for its implication in CT74,75. Each antigen’s coding sequences were synthesized and fused to a C-terminal 6 histidine-tag inserted into a pET28a + expression vector (Genscript, Piscataway, Netherlands). The transformation was carried out using competent SHuffle bacteria (T7 E. coli) for the expression of PfAMA1 (24-546aa) and TgAMA1 (67-484aa), and E. coli BL21C + for the expression of TgSAG1 (46–336 aa) and TgGRA7 (27-183aa). Our recombinant proteins were produced in full length, excluding only the signal sequences and the transmembrane domains associated with intracellular peptides as annotated by UniProt.
The bacterial transformation was performed following the Neb Bioland protocol described on the website (https://www.neb.com/en/protocols/2012/05/21/transformation-protocol). Transformed bacteria were spread on a Petri dish containing LB agar (Sigma Aldrich, St-Quentin-Fallavier, France) supplemented with 25 µg/ml of kanamycin (Gibco, United Kingdom). The plate was then incubated for 24 h at 30°C for transformed SHuffle bacteria and 37°C for BL21C + to allow bacterial growth and selection.
Bacterial culture and induction
A single colony from the selection plate was transferred to 25 mL of Luria Broth medium (Invitrogen™, France) supplemented with 25 µg/ml of kanamycin. The pre-culture was incubated overnight at 30 °C for SHuffle bacteria and 37°C for BL21C+. Bacteria were then cultured in 1 L of Luria Broth medium containing 25–50 µg/ml of kanamycin, with a starting optical density (OD) of 0.1. Protein expression was induced for 3 h by adding 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, St-Quentin-Fallavier, France) when the bacterial growth reached an OD between 0.6 and 0.8. The bacterial pellet was collected by centrifugation at 4000 rpm for 30 min and stored at −20°C for later use.
An SDS-PAGE gel (Bio-Rad, France) was performed to confirm protein induction at the expected molecular weight for each antigen i.e. 75 kDa for PfAMA1, 50 kDa for TgAMA1, 30 kDa for TgSAG1 and 25 kDa for TgGRA7 (Supp. Figure 3 and Supp. Figure 4).
Purification of PfAMA1, TgAMA1 and TgSAG1 recombinant proteins
The bacterial cells were lysed in phosphate buffer saline (Gibco™ PBS, Fisher Scientific, France) lysis buffer with the NaCl concentration adjusted at 350 mM, supplemented with 1 tablet of protease inhibitor cocktail (Roche cOmplete™ EDTA free), 5 mM DTT and 100 mM of lysozyme (all products from Sigma Aldrich, St-Quentin-Fallavier, France). The suspension was incubated on ice for 15 min and then sonicated six times alternating 10-second cycles of sonication and rest, to disrupt cell membranes and release the proteins expressed in inclusion body (IB) forms. The sonicated lysate was centrifuged at 6000 rpm, at 4°C for 30 min. The supernatant was discarded and the pellet, containing the IB, was washed twice with lysis buffer supplemented with 4 M urea (Fisher Scientific, France) to remove impurities. The IB were then dissolved overnight at 4°C in a buffer at pH 8 containing PBS 1X, 1% Triton X-100 (Fisher Scientific, France) and 6 M urea. The dissolved mixture was centrifuged at 9000 rpm for 30 min at 4°C to remove insoluble debris. The solubilized proteins were incubated with Ni-NTA agarose resin (Fisher Scientific, France) at 4°C for 1.5 h for metal affinity purification of the 6xHis-tagged recombinant fusion proteins. The protein refolding was initiated by gradually reducing the urea concentration from 6 M to 4 M and 2 M until reaching a final concentration of 1 M.
The PfAMA1 recombinant protein was refolded on Ni-NTA column in PBS 1X buffer at pH 8.5, supplemented with 5 mM of reduced glutathione GSH and 0.5 mM of oxidized glutathione GSSG (Sigma Aldrich, St-Quentin-Fallavier, France) for 2 h at 4°C. The refolded protein was then eluted in PBS 1X with 400 Mm of imidazole (Arcos Organics, Geel, Belgium). In order to remove the residual contaminants, the eluate was filtrated in size chromatography by injecting 5 mL of the eluted protein into the gel filtration column (HiLoad® 16/600 Superdex® 200, Cytiva) connected to an Äkta purifier system (AKTA pure™, Cytiva).
The TgAMA1 and TgSAG1 recombinant proteins were eluted in PBS 1X with 1 M urea and 400 mM imidazole. The proteins were then dialyzed against a 10 kDa cut-off membrane (Slide-A-Lyzer™ G3 Dialysis Cassettes, Fisher Scientific, France) for 24 h in PBS 1X buffer at pH 8.5, supplemented with 3 mM GSH and 0.3 mM GSSG.
The concentrations of the three purified proteins were measured at 280 nm using a Nanodrop and aliquots were stored in glycerol 10% at −80°C for long-term preservation.
Purification of the soluble TgGRA7 Recombinant protein
The soluble GRA7 protein was directly purified on the Ni-NTA resin from the supernatant obtained after the lysis of the bacterial cells. The protein was subsequently eluted from the nickel column using 400 mM imidazole after three washes with PBS 1X containing 20 mM imidazole. Gel filtration was then performed to remove E. coli contaminants. The eluted protein was concentrated before being injected into a gel filtration column connected to an Äkta system. The protein concentration was measured at 280 nm using a Nanodrop and aliquots were stored in 15% of glycerol at −80°C for long-term preservation.
Pfs48/45 Recombinant antigen
The gametocyte recombinant antigen Pfs48/45 was produced and kindly gifted by Michael Theisen laboratory research76 (Department for Congenital Disorders, Statens Serum Institut, Copenhagen, Denmark).
Titration of specific IgG directed to PfAMA1, TgAMA1 TgSAG1 and TgGRA7 with an enzyme-linked immunosorbent assay (ELISA)
Nunc MaxiSorp 96-well microtiter plates (Thermo Scientific™, France) were coated with purified IgG sourced from human serum (Sigma Aldrich, St-Quentin-Fallavier, France) at concentrations ranging from 5000 to 10 ng/ml, in PBS 1X coating buffer at pH 8.5. Additionally, recombinant antigens were individually coated at a concentration of 1.5 µg/ml for PfAMA1, TgAMA1, TgSAG1 and TgGRA7 and of 0.5 µg/ml for Pfs48/45 in coating buffer, with 100 µl added per well, and left to incubate at 4 °C overnight. Plasma samples were appropriately diluted (1:5000 for PfAMA1 and 1:100 for TgAMA1, TgSAG1, TgGRA7 and Pfs48/45 antigens) in dilution buffer [PBS 1X − 0.5% Tween 20 − 1% BSA (Sigma Aldrich, St-Quentin-Fallavier, France) − 0.02% sodium azide (Sigma Aldrich, St-Quentin-Fallavier, France)]. Following the dilution, the coated plates underwent three washes of 1 min each with washing buffer [PBS 1X − 0.5% Tween 20 − 0.5 M NaCl]. Subsequently, each well received 150 µl of blocking buffer [PBS 1X − 0.5%Tween 20 − 3% BSA], and the plates were then incubated at room temperature for 1 h. The diluted plasma samples (100 µl each) were added in duplicate to the coated plates containing the recombinant antigens, and then incubated for 1 h. After another round of washing, a primary antibody (mouse anti-human Fc, Sigma Aldrich, Schnelldorf, Germany) diluted at 1:4000 in dilution buffer was applied and left to incubate for 1 h at room temperature on a rocking platform, followed by three washes with washing buffer. Finally, a secondary antibody (peroxidase-conjugated goat anti-human IgG H + L, Bio Rad, France) was added at 1:6000 in dilution buffer. After the last wash, 100 µl of 3,3′,5,5′-Tetramethylbenzidine (TMB, Sigma Aldrich, St-Quentin-Fallavier, France) were added to each well and incubated for 30 min at room temperature in the dark. The reaction was stopped by adding an equal volume of 0.2 M sulfuric acid (H2SO4, Sigma Aldrich, St-Quentin-Fallavier, France). Absorbance was read at 450 nm with a reference wavelength of 620 nm using a plate reader (TECAN Infinite 50). The concentration of specific antibodies was calculated using MyAssay Software (https://www.myassays.com/) using a five-parameter logistic modelisation. For each antigen, the cut-off was represented by calculating the mean + 2 SD of the values obtained in the group of 15 malaria non-exposed and toxoplasmosis seronegative plasma samples from the TOXODIAG study.
In vitro growth inhibition assay (GIA) of merozoites from the 3D7 strain of P. falciparum
To assess the P.f inhibitory activity of the anti-T.g antibodies contained in the plasma samples, an in vitro culture of the P.f 3D7 strain was maintained with O + red blood cells from healthy donors at a 5% haematocrit in T75 sterile culture flasks containing 25 ml of RPMI 1640 medium (Gibco™, Fisher Scientific, France) – 25 mM HEPES (Gibco™, Fisher Scientific, France) at pH 7.4 supplemented with 20 mg/ml hypoxanthine (Gibco™, Fisher Scientific, France), 25 mM NaHCO3 (Fisher Scientific, France), 2.5 mg/ml gentamycin (Gibco™, Fisher Scientific, France), 0.5% Albumax II (Gibco™, Fisher Scientific, France) and 10% human AB serum (Biowest, France) in an atmosphere of 5% O2, 5% CO2 and 90% N2. The culture medium was changed every 24 h while maintaining a parasitemia below 5%. Thin blood smears were made to verify the parasite stages and calculate parasitemia. Parasite stages were synchronized and parasites were concentrated using a magnetic activated cell sorter (MACS) column (Miltenyi Biotec, Germany) to collect schizont forms. These mature forms were diluted at a 2% haematocrit in the culture media previously described, at an initial parasitaemia of 1% for one-cycle assays. For multi-plate assays, a malaria-positive control plasma (PC) selected from the PDC group with a high inhibition activity and seronegative for toxoplasmosis was included in each plate along with a pool of negative control plasmas (NC) collected from 10 French donors non-immune against malaria and toxoplasmosis (National Malaria Reference Center, Bichat – Claude Bernard Hospital). The assay setup involved adding 100 µl of the suspension of P.f 3D7 mature forms to each well of 96-well culture plates, adjusting the plasma dilution to 1:50 final concentration. Each sample was tested in triplicate. The plates were maintained in a specialized culture chamber fully gassed at 5% CO2 before incubation at 37 °C and 90% humidity for 48 h, corresponding to 1 parasite replication cycle. Parasitemia was measured using SYBR Green 1X (Sigma Aldrich, St-Quentin-Fallavier, France) after facilitating cell membrane disruption with lysis buffer [20 mM Tris (Sigma Aldrich, St-Quentin-Fallavier, France), 5 mM EDTA (Sigma Aldrich, St-Quentin-Fallavier, France), 0.008% (w/v) saponin (Sigma Aldrich, St-Quentin-Fallavier, France) and 0.08% (v/v) Triton X-100]. The fluorescence absorbance was measured using the microplate SAFAS XENIUS reader (SAFAS Monaco, Monaco) with an excitation wavelength of 480 nm and an emission at 520 nm. Data analysis involved calculating the percentage of inhibition based on OD reading using the formula: [100 – (OD test plasma parasitemia/OD non-immune plasma parasitemia × 100)]. In order to reduce inter-plate variation, normalization factors were applied to the percentage of inhibition using the formula [(ODm PC) – (ODm NC)] / [(OD PC) – (OD NC)] where ODm represents the mean reactivity of PC and NC for all plates, and OD PC and OD NC are the mean reactivities of the control plasma samples for a specific plate.
Statistical analyses
A quantitative comparison of the P.f and T.g antibody responses was made with a one-way analysis of variance (ANOVA) in multiple comparisons using a Tukey correction. A Pearson correlation was carried out in order to evaluate the linear correlation between P.f and T.g antibody responses in the case of the malaria-exposed population groups (PDC and COA). Graphics were generated using the software GraphPad Prism version 10.
Linear or logistic regressions were conducted in univariate analyses to identify significant predictors (with p ≤ 0.20) to be included in subsequent step-by step multivariate analyses. The statistical analyses comprised various regression models to identify, in each population group, independent variables associated with either functional antibody response (GIA), positive toxoplasmosis serology or positive malaria serology. An interaction between P.f and T.g antibody response variables was added to evaluate their combined effects in the final multivariate model. Analyses were performed using Stata version 13 (StatCorp LP, TX, USA). Statistical significance was set at p ≤ 0.05.
Ethics approval
The TOXODIAG study “New diagnostic approach for congenital toxoplasmosis” received the ethical clearance of the Comité de Protection des Personnes (CPP) Sud-Méditerranée II from France on October 3, 2017, under the ID RCB 2017-A02208-45. The CoaLa study “Caractérisation de la production d’immunOglobulines spécifiques d’antigènes pArasitaires par les Lymphocytes B néonAtaux dans le cadre d’infections congénitales” was authorized by the Comité National d’Ethique pour la Recherche en Santé́ (CNERS) from Benin, with the n°40 assigned on September 28, 2017. Similarly, the PlasDCty study “Elucider et contourner la tolérance immunitaire au paludisme” was authorized by the CNERS under the n°4 allocated on March 23, 2021. For each study, enrolled women signed an informed written consent before their inclusion and approved the use of the collected data. They were explicitly given the choice to withdraw from each study at any stage. Each study followed the WMA Declaration of Helsinki, adhering to ethical principles for medical research involving human subjects. All the methods were performed in accordance with the approved guidelines and regulations.
