B-cell and T-cell epitope prediction
Fine epitope mapping through bioinformatics
The predicted entire MAEBL protein sequence of the rodent malaria parasite P. yoelii, available at PlasmoDB (PYYM_0902200.1) was initially used to predict C57BL/6 J mouse MHC epitopes for H-2Kb, H-2Db (MHC class I) and IA-b alleles (MHC class II). Predictions for linear B lymphocyte epitopes were also ran. Detailed procedures were as follows.
MHC class I and II epitope mapping
Rankpep software [13,14,15,16] set to a binding threshold of 3% and proteasome cleavage filter “ON” was used for initial MHC class I epitope screening. Subsequently, the MAEBL protein was re-screened using IEDB [17, 18], NetMHCpan [19, 20], Bimas [21, 22], MAPPP [23, 24] and PropredI [25]. All epitopes that were identified using all softwares were thus selected as those presenting the highest confidence.
Rankpep was also used to predicted MHC class II epitopes, with a binding threshold set to 3% and proteasome cleavage filter “OFF”. Resulting predicted epitopes with a score above 9.52 was selected for further analyses using IEDB [17, 18] and NetMHCII [26,27,28]. Epitopes displaying the best scores generated between all screens were included in the final list.
B-cell epitope mapping
The BCPRED resource [29, 30] was employed to identify B-cell epitopes with a size of 20 aminoacids, set to a 75% specificity. After identifying the best predicted epitopes based on score, each of these epitopes was screened for predicted antigenicity using the VaxiJen v.2 software [31], under a 0.5 threshold and the “parasite model” filter on, according to previously published recommendations [32, 33]. Subsequently, all epitopes presenting scores above 0.5 were included in the final list. Lastly, the potential conservation within each of these epitopes between P. yoelii and P. falciparum, and between P. yoelii and P. vivax was investigated, using the BLAST tool at PlasmoDB, and epitopes were considered homologous between P. yoelii and any of the other two human parasites when amino acid identity was higher than 50%.
Immunization regimen
C57BL/6 mice with 5–7 weeks-old were injected subcutaneously four times at 3 weeks intervals with 5 μg of rPyM2-MAEBL emulsified in 1:1 (vol/vol) complete Freund’s adjuvant (CFA) for the first dose or incomplete Freund’s adjuvant (IFA) in the subsequent doses [4].
The prime-boost (PB) group received the first dose of 100 μg pIgSPM2 intramuscular, followed by three doses of 5 μg of rPyM2-MAEBL in IFA. As control groups, animals were injected with 1:1 (vol/vol) adjuvant (CFA/IFA), pIgSPM2 or only pIgSP vector. Sera from immunized mice were collected immediately before each dose and 3 weeks after the last dose. There was no significantly difference in protection between the rM2-MAEBL and the prime-boost regimen. All experiments and procedures were performed in accordance with relevant guidelines and regulations of the Ethical Committee for Animal Research of the University of Campinas and were approved under Protocol No. 1437-1.
Slide preparation and immunofluorescence assays (IFA)
Clinical isolates of P. vivax and P. falciparum infected blood from malaria patients were collected at Shoklo Malaria Research Unit (Thailand) with written informed consent. The thin smears used for the IFA were prepared from ex vivo matured schizonts concentrated by 45% Percoll for P. vivax and 70% Percoll for P. falciparum [3, 34] that were diluted 1:4 with uninfected RBCs. Immunofluorescence assays were performed after fixing the blood smears with ice-cold acetone for 20 min and air-dried. Well diameters were established with the aid of a Dako-Pen (Dako), and blocking was performed by 30-min incubation at 37 °C with PBS containing 3% BSA (USB). C57BL/6 mice with 5–7 weeks-old were immunized as described elsewhere [4] and pooled sera from the different immunization groups were diluted 1:50 in PBS supplemented with 3% BSA and applied to the slides for 1 h at 37 °C. Slides were washed 3X in PBS and incubated with Alexa-568 goat anti-mouse IgG (Invitrogen) for 1 h at 37 °C in the dark, then washed 3X in PBS and incubated with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride-(Invitrogen) diluted in ultrapure (Millipore) water for 10 min at room temperature. After another round of washing, Fluorosave (Caliobiochem) was added, and slides were sealed with coverslips. Parasites were visualized with the aid of a Nikon TS100 epifluorescence microscope. All samples above were collected in accordance with relevant ethical guidelines and regulations of the University of Oxford, Centre for Clinical Vaccinology and Tropical Medicine and the Ethics committee of Faculty of Tropical Medicine, Mahidol University, under the approved protocols OXTREC 027-025 and MUTM 2008-215 from.
Plasmodium-specific antibody binding assay
Transfection
Transfection of HEK293 cells to obtain cells expressing PvMAEBL, PfMAEBL, PfSEA or PVX_113775 on the cell surface was as previously described [35]. Nucleotide sequences encoding for PvMAEBL (PVX_092975: amino acid 540–1007), PVX_113775 (amino acid 1–358), PfMAEBL (PF3D7_1147800: amino acid 958–1249) and PfSEA (PF3D7_1021800: amino acid 810–1083) were amplified via PCR, using either P. vivax UMS203 or P. falciparum 3D7 RNA as template, and cloned into the pDisplay vector (Invitrogen). The resultant plasmids were then transfected into HEK293 cells using lipofectamine 2000 (Invitrogen) for surface expression of the antigens.
Antibody binding assay
The antibody binding assay was as previously described [35]. Briefly, transfected cells, expressing the antigens on the cell surface, were first incubated with the serum (diluted 1:100 in FACS blocking buffer (10% FBS in PBS) on shaking. The cells were then incubated with a double stain, consisting of Alexa Fluor 488-coupled secondary antibodies (Invitrogen; diluted 1:500) and propidium iodide (PI; diluted 1:2500) on shaking. Cells were read on Accuri C6 (BD Biosciences) and analysed using FlowJo (Tree Star).
Ex-vivo functional invasion assays
Plasmodium falciparum isolates
The preparation of schizonts concentrate after 70% Percoll was mixed with target cells (RBCs/uninfected erythrocytes) in the ratio 1:12. The solution was diluted with 2% haematocrit using McCoy 5A 10% human AB serum (inactivated) and cultivated in microplates in a volume of 200 µl, 5% O2 at 37.5° C for 12 h. The inhibitory potential of anti-PyM2-MAEBL antibodies were tested by adding the fourth pool of sera from animals immunized with a dose of the rPyM2-MAEBL protein alone or in heterologous prime-boost system at the 1:50 dilution to the final mixture invasion assay. It was used as a control serum pool of the 4th dose animals of ACF/AIF group. As positive control, it was used 100 μM of E64 (Sigma), a protease inhibitor, to ensure no disruption of schizonts. At the end of the test were made blood smear slides of each well stained with Giemsa (Sigma). The number of rings/trophozoites to 1000 cells was determined by microscopic analysis.
Plasmodium vivax isolates
20 ml of cord blood were collected in heparin tube immediately after the newborn delivery. Samples were collected after informed consent was obtained from each subject and in accordance with relevant guidelines and regulations of the ethics committee of the Fundação de Medicina Tropical—Dr. Heitor Vieira Dourado (protocol CAAE-0044.0.114.000-11).
The blood group was determined by using a standard ABO Kit (EBRAN). After plasma removal, the cells were washed in McCoy 5A medium (Sigma) and haematocrit adjusted to 50% using McCoy 5A. Leukocytes and platelets were depleted by two passages in CF11 filtration column (Whatman). Red cells from umbilical cord were again adjusted to 50% haematocrit and carefully overlaid on tubes containing 70% Percoll (GE Healthcare). According to Russell et al. [3], the preparation of schizonts concentrate after 45% Percoll enrichment was mixed with target cells (cord blood) in the proportion 1: 6. The solution was diluted to 2% haematocrit using McCoy 5A (Sigma) 20% human AB serum (inactivated) and cultivated in microplates in a volume of 200 µl 5% O2 at 37.5 °C for 24–30 h depending on the parasite maturation. The inhibitory potential of anti-PyM2-MAEBL antibodies were tested by adding the fourth dose pool sera from animals immunized with rPyM2-MAEBL protein alone or in heterologous prime-boost system at the 1:50 dilution to the final mixture invasion assay. It was used as a control sera pool of the 4th dose animals of ACF/AIF group. As a positive control of inhibition was used polyclonal anti-P. vivax Duffy Binding Protein (PvDBP) IgG [36]. At the end of the test blood smear slides of each well were performed and stained with Giemsa (Sigma). The number of rings/trophozoites from 1000 cells was determined by microscopic analysis.
Amplification and maebl sequencing from Amazonian isolates
A total amount of nine blood samples were collected (during the period of 2012–2014) from different areas in the Amazon: the cities of Manaus in the Amazonas state and Mâncio Lima and Acrelândia, both in state of Acre (Fig. 1). The identification of P. vivax species was performed by nested-PCR as previously described [37]. For Pv-MAEBL amplification, oligonucleotides were designed based on the Pv-MAEBL Sal-1 strain (PVX_092975) sequence from PlasmoDB. Three DNA fragments were PCR-amplified to obtain the sequence of M2-MAEBL domain. Oligonucleotide sequences used in this study are displayed in Additional file 1. Reactions to amplify fragments one and three were performed with Platinum Taq (Invitrogen). Amplification condition was as follows: 1 cycle of 5 min at 95 °C and 35 cycles of 30 s at 95 °C, 45 s at 58 °C, 1 min at 72 °C and a final cycle of 5 min at 72 °C. The fragment two was amplified using Phusion DNA Polymerases (Thermo Fisher Scientific). The PCR reaction was submitted to 1 cycle of 5 min at 98 °C and 35 cycles of 15 s at 98 °C, 30 s at 59 °C, 1 min at 72 °C and a final cycle of 10 min at 72 °C. The purified PCR product was sequenced using 3730 × l DNA Analyzer (Applied Biosystems). All generated sequences were subjected to similarity search analysis by BLAST.
Alignment of the M2 maebl domain sequences of P. vivax
The MAEBL M2 domain of the nine isolates from Brazilian Amazon (GenBank accession nos. KX061004 to KX061012) were compared to previously described sequences deposited in PlasmoDB: Thailand_VKBT-101, Thailand_VKBT-100, Thailand_VKTS-39 and the reference sequence Pv-MAEBL Sal-1 strain (PVX_092975), using the Clustal Multialin Interface Page [38].