In silico epitope mapping and experimental evaluation of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) as a malaria vaccine candidate
- Pedro Cravo1, 2, 3Email author,
- Renato B. Machado2,
- Juliana A. Leite4,
- Taizy Leda2,
- Rossarin Suwanarusk5,
- Najara Bittencourt4,
- Letusa Albrecht4, 6,
- Carla Judice4,
- Stefanie C. P. Lopes4, 7,
- Marcus V. G. Lacerda7, 8,
- Marcelo U. Ferreira9,
- Irene S. Soares10,
- Yun Shan Goh5,
- Daniel Y. Bargieri9,
- François Nosten11,
- Bruce Russell12,
- Laurent Rénia5 and
- Fabio T. M. Costa4
© The Author(s) 2018
Received: 24 February 2017
Accepted: 18 December 2017
Published: 10 January 2018
Technical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated.
Detailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity.
The MAEBL antigen is promising candidate towards the development of a malaria vaccine.
Malaria is one of the most nefarious infectious diseases of humans and continues to have a devastating global impact upon health and well-being, mainly among children under the age of five and pregnant women. Annually, about 200 million cases are reported and almost 600,000 deaths occur . Despite the recent relative progress towards a Plasmodium falciparum vaccine, the development of a vaccine to protect individuals from Plasmodium vivax is still incipient, jeopardizing the Malaria Vaccine Roadmap , and consequently the whole malaria eradication agenda. Currently, the difficulty to grow P. vivax in vitro for long periods  challenges ongoing strategies for identification of novel vaccine candidates against this parasite. Considering these roadblocks, an approach to develop vaccines against P. vivax might rely on the use of malaria antigens conserved amongst species.
Recently, the immunogenicity of M2 MAEBL (Merozoite Adhesive Erythrocytic Binding Protein) domain of Plasmodium yoelii has been demonstrated. This vaccine candidate conferred 90% protection in immunized mice after lethal challenge and corresponding antisera inhibited significantly erythrocyte invasion by P. yoelii . MAEBL is a membrane protein that belongs to the erythrocyte binding protein (ebl) family [5–8] and exhibits homologous regions to the DBL-EBP (Duffy binding like–Erythrocyte binding protein) and to the apical membrane antigen 1 (AMA1) [7–9]. MAEBL is a conserved antigen expressed in blood-stage late forms, in the salivary gland sporozoite and also during the late liver stage [10, 11].
Immunoinformatics, in which computational approaches select the most appropriate vaccine candidate and algorithms predict T- and B-cell immune epitopes , is an alternative to overcome limitations imposed by the lack of knowledge in terms of P. vivax biology.
Here, an immunoinformatics strategy was used, that identified the MAEBL antigen as a promising interspecies and interstrain malaria vaccine candidate and the pan-reactivity of the P. yoelii M2-MAEBL antisera against P. falciparum and P. vivax was investigated.
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 [13–16] 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 . 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 [26–28]. 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 , 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%.
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 .
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  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 of HEK293 cells to obtain cells expressing PvMAEBL, PfMAEBL, PfSEA or PVX_113775 on the cell surface was as previously described . 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 . 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. , 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 . 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
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 .
Detailed in silico epitope mapping of the P. yoelii Merozoite Adhesive Erythrocytic Binding Protein (MAEBL)
MHC class I and II epitope mapping
In silico epitope mapping identified two predicted epitopes for MHC class I, for each of the H-2Kb and H-2Db alleles (Additional file 2). Epitope QNYYSFTNL for the H-2Kb allele presented the highest score between all programs used, whereas the NQNINLVKL epitope for allele H-2Db was among the top ten with the highest scores. Initial epitope screening for MHC class II using Rankpep resulted in the identification of 19 epitopes. However, after further analysis with IEDB and NetMHCII, only two epitopes were common between all softwares (Additional file 2).
B-cell epitope mapping
Immunofluorescence and invasion inhibition assays with P. falciparum and P. vivax isolates
Alignment of the M2 maebl domain sequences
The M2 maebl domain of P. vivax of nine patients from three different areas in the Amazon was sequenced (Additional file 4). The isolates were compared to three Thai isolates as well as the reference strain Sal-1 sequences previously deposited in PlasmoDB. A high degree of similarity was observed among all sequences, with only two SNPs differentiating the Brazilian and Sal-1 sequences from Thai parasites (Additional file 4). These findings may largely explain the similar inhibitory levels observed in functional assays amongst different isolates and reinforce the validity of using the P. vivax Sal-1 strain genome available at PlasmoDB for immunoinformatics analysis.
Functional assays from previous work have suggested that MAEBL is a suitable malaria vaccine candidate . For this reason, MAEBL was studied in further detail here, through a conjugation of detailed epitope mapping in silico and further experimental evaluation. Epitope mapping identified four MHC classes I and II putative epitopes within the P. yoelii MAEBL antigen, which resulted from a consensus between different prediction programs. It has been shown that MAEBL is expressed not only in blood stages, but also in midgut and salivary gland sporozoites [11, 40, 41] suggesting that it may be important for the establishment of a successful pre-erythrocytic infection of the parasite. As such, in light of the discovery of a malaria vaccine, selecting antigens that present MHC class I and II epitopes should be prioritized given the mounting evidence that antigen-specific CD8+ and CD4+ immune responses contribute for protection from sporozoite challenge both in animal models and human malaria . In addition, it was determined that the MAEBL antigen contains at least 25 predicted B-cell epitopes that are likely to elicit antibody-dependent immune responses, which have been widely shown to be important for protection against blood-stage antigens . Although these epitopes were scattered throughout different domains of MAEBL, more than half of them (fourteen) are located within the M1 and M2 ligand domains, which have been confirmed to bind to mouse erythrocytes , suggesting their role in cell invasion. Moreover, all but one of these fourteen epitopes lying in the M1 and M2 domains are conserved between P. yoelii, P. vivax and P. falciparum. It has been shown that a particular epitope (YVSSFIRPDYETKCPPRYPL) present in the P. falciparum MAEBL M2 domain is highly immunogenic and capable of binding human HLA with high avidity . Interestingly, the present bioinformatics predictions identified an epitope with a very similar sequence (SSFIRPDYETKCPPRYPL) that is conserved between P. yoelii, P. falciparum and P. vivax. Since the M2 peptide was used here in the immunization experiments, the extant conservation of this and the other epitopes may largely explain the pan-reactivity observed and further suggests that this peptide may be a promising candidate for a malaria vaccine targeting more than one parasite species. In fact, previous experimental MAEBL epitope mapping revealed that anti-MAEBL IgM and IgG antibodies displayed strong responses against fragments located in the M2 region .
Plasmodium merozoites may use multiple pathways to invade red blood cells. The latter point is supported by data from previous experiments in which MAEBL knockout Plasmodium berghei strain ANKA parasites were still able to invade red blood cells in vivo . Although erythrocyte invasion by Plasmodium merozoites seems to be largely host-specific, different Plasmodium species keep several structurally and functionally homologous adhesins that play important roles during invasion, which enable them to, in some cases, invade red cells of heterologous host species . It was shown that proteins from P. falciparum and P. yoelii bound to heterologous host red cell receptors, likely through their conserved motifs; suggesting homologies between Plasmodium ligands . Bioinformatics analyses show high similarities between the functional and genomic sequences across Plasmodium species [45, 46]. Also, studies suggest preservation of erythrocyte binding function of phylogenetically distant Plasmodium species . These observations indicate that interspecies conserved epitopes that map within conserved functional motifs could be promising candidates for the development of effective vaccines.
MAEBL is an antigen expressed during the blood and liver stages and is essential for sporozoite invasion of mosquito salivary glands . MAEBL is also implicated in the invasion of merozoites into new erythrocytes, though not essential at this stage. The ability of anti-MAEBL antibodies to inhibit the invasion of merozoites of P. falciparum was demonstrated here. Considering that MAEBL seems to be a highly conserved antigen and expressed at different parasite stages, determination of its potential as a vaccine is paramount. Indeed, it has recently been shown that M2 MAEBL immunization confers 90% mice survival against lethal challenge with P. yoelii parasites .
Furthermore, the pan-reactivity of the anti-sera raised against rPyM2-MAEBL was revealed, which is based on the M2 domain of the P. yoelii MAEBL antigen. In addition, the fact that HEK cells expressing the PvMAEBL and the PfMAEBL showed reactivity presents a counter proof of the pan-reactivity of the anti-sera raised against the PyMAEBL M2 region and thus validated the ex vivo assays. An IFA pattern different from the article published by Leite et al. Was detected . Here, a cytosolic staining was observed and previously an apical staining was described. This difference could be justified by the fact that the anti-serum could recognize different Plasmodium proteins. These antibodies were able to recognize and inhibit at high levels the invasion of P. falciparum and P. vivax (mainly) collected from infected patients in Thailand, suggesting that antibodies against one Plasmodium species recognize several antigens from other species [34, 47, 48] and the presence of homologous molecules with interspecies conserved epitopes.
Combined immunoinformatics and experimental approach strategies confirmed the potential of the MAEBL antigen as a malaria vaccine candidate. Whilst the MAEBL may be unlikely to confer sterile immunity if used as the sole component of a vaccine, it may be combined with other antigens towards the synthesis of a fully effective chimeric subunit vaccine. Moreover, the P. yoelii mouse model can be used to test human malaria parasite antigens for their immunogenicity and efficacy as vaccine candidates.
PC, RBM, TL and LA performed the immunoinformatics and sequence analyses. JAL, RS, NB, CJ, SCPL, BR and YSG carried out laboratory work. MVGL, DYB, MUF, ISS, FN and LR participated in the data analyses and helped to draft the manuscript. PC and FTMC conceived the study and wrote the final version of the manuscript. All authors read and approved the final manucsript.
We would like to thank Professor Francois Nosten (Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Thailand) for providing the P. vivax UMS203 infected red blood cells which were obtained from malaria patients at Shoklo Malaria Research Unit, Tak province, Thailand.
The authors declare they have no competing interests.
Availability of data and materials
All data and materials are available upon request.
Consent for publication
Ethics approval and consent to participate
All animal 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.
Clinical isolates of P. vivax and P. falciparum infected blood from malaria Thai patients were collected at Shoklo Malaria Research Unit (Thailand) with written informed consent. 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.
Samples from patients in the Amazon 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).
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP—Grant # 2012/16525-2), Instituto Nacional de Ciência e Tecnologia de Vacinas (INCTV) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). RBM was supported by a CAPES fellowship. JAL and NB were sponsored by FAPESP fellowships. CC was supported by a CNPq fellowship. PC, MVGL, MUF and FTMC are CNPq research fellows in productivity. This study and RS and BR received funding from SIgN under the Agency for Science, Technology and Research (A*STAR, Singapore).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- World Health Organization. World malaria report 2013. Geneva: World Health Organization; 2013.Google Scholar
- Malaria Vaccine Funders Group. Malaria vaccine technology roadmap. Geneva: World Health Organization; 2013.Google Scholar
- Russell B, Suwanarusk R, Borlon C, Costa FT, Chu CS, Rijken MJ, et al. A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax. Blood. 2011;118:e74–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Leite JA, Bargieri DY, Carvalho BO, Albrecht L, Lopes SC, Kayano AC, et al. Immunization with the MAEBL M2 domain protects against lethal Plasmodium yoelii infection. Infect Immun. 2015;83:3781–92.View ArticlePubMedPubMed CentralGoogle Scholar
- Blair PL, Kappe SH, Maciel JE, Balu B, Adams JH. Plasmodium falciparum MAEBL is a unique member of the ebl family. Mol Biochem Parasitol. 2002;122:35–44.View ArticlePubMedGoogle Scholar
- Kappe SH, Curley GP, Noe AR, Dalton JP, Adams JH. Erythrocyte binding protein homologues of rodent malaria parasites. Mol Biochem Parasitol. 1997;89:137–48.View ArticlePubMedGoogle Scholar
- Kappe SH, Noe AR, Fraser TS, Blair PL, Adams JH. A family of chimeric erythrocyte binding proteins of malaria parasites. Proc Natl Acad Sci USA. 1998;95:1230–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Noe AR, Adams JH. Plasmodium yoelii YM MAEBL protein is coexpressed and colocalizes with rhoptry proteins. Mol Biochem Parasitol. 1998;96:27–35.View ArticlePubMedGoogle Scholar
- Michon P, Stevens JR, Kaneko O, Adams JH. Evolutionary relationships of conserved cysteine-rich motifs in adhesive molecules of malaria parasites. Mol Biol Evol. 2002;19:1128–42.View ArticlePubMedGoogle Scholar
- Preiser P, Rénia L, Singh N, Balu B, Jarra W, Voza T, et al. Antibodies against MAEBL ligand domains M1 and M2 inhibit sporozoite development in vitro. Infect Immun. 2004;72:3604–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Kariu T, Yuda M, Yano K, Chinzei Y. MAEBL is essential for malarial sporozoite infection of the mosquito salivary gland. J Exp Med. 2002;195:1317–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Heinson AI, Woelk CH, Newell ML. The promise of reverse vaccinology. Int Health. 2015;7:85–9.View ArticlePubMedGoogle Scholar
- Reche PA, Glutting JP, Reinherz EL. Prediction of MHC class I binding peptides using profile motifs. Hum Immunol. 2002;63:701–9.View ArticlePubMedGoogle Scholar
- Reche PA, Glutting JP, Zhang H, Reinherz EL. Enhancement to the RANKPEP resource for the prediction of peptide binding to MHC molecules using profiles. Immunogenetics. 2004;56:405–19.View ArticlePubMedGoogle Scholar
- Reche PA, Reinherz EL. Prediction of peptide-MHC binding using profiles. Methods Mol Biol. 2007;409:185–200.View ArticlePubMedGoogle Scholar
- Rankpep. Prediction of binding peptides to Class I and Class II MHC molecules. http://imed.med.ucm.es/Tools/rankpep.html. Accessed 01 June 2016.
- IEBD.org. Free epitope database and prediction resource. http://www.iedb.org/. Accessed 30 June 2016.
- Vita R, Overton JA, Greenbaum JA, Ponomarenko J, Clark JD, Cantrell JR, et al. The immune epitope database (IEDB) 3.0. Nucleic Acids Res. 2015;43:D405–12.View ArticlePubMedGoogle Scholar
- Hoof I, Peters B, Sidney J, Pedersen LE, Sette A, Lund O, et al. NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics. 2009;61:1–13.View ArticlePubMedGoogle Scholar
- NetMHCpan 2.4 Server. http://www.cbs.dtu.dk/services/NetMHCpan-2.4/. Accessed 10 july 2016.
- HLA peptide binding predictions. https://www-bimas.cit.nih.gov/molbio/hla_bind/. Accessed 30 July 2016.
- Parker KC, Bednarek MA, Coligan JE. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol. 1994;152:163–75.PubMedGoogle Scholar
- Hakenberg J, Nussbaum AK, Schild H, Rammensee HG, Kuttler C, Holzhütter HG, et al. MAPPP: MHC class I antigenic peptide processing prediction. Appl Bioinform. 2003;2:155–8.Google Scholar
- MAPPP. MHC-I antigenic peptide processing prediction. http://www.mpiib-berlin.mpg.de/MAPPP/. Accessed 08 Aug 2016.
- Singh H, Raghava GP. ProPred1: prediction of promiscuous MHC Class-I binding sites. Bioinformatics. 2003;19:1009–14.View ArticlePubMedGoogle Scholar
- NetMHCIIpan 3.1 Server. http://www.cbs.dtu.dk/services/NetMHCIIpan/. Accessed 12 Aug 2016.
- Nielsen M, Lundegaard C, Lund O. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method. BMC Bioinform. 2007;8:238.View ArticleGoogle Scholar
- Nielsen M, Lund O. NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinform. 2009;10:296.View ArticleGoogle Scholar
- BCPREDS. B-cell epitope prediction server. http://ailab.ist.psu.edu/bcpred/. Accessed 25 Aug 2016.
- El-Manzalawy Y, Dobbs D, Honavar V. Predicting linear B-cell epitopes using string kernels. J Mol Recognit. 2008;21:243–55.View ArticlePubMedPubMed CentralGoogle Scholar
- VaxiJen. Prediction of Protective Antigens and Subunit Vaccines. http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html. Accessed 03 Sept 2016.
- Doytchinova IA, Flower DR. Identifying candidate subunit vaccines using an alignment-independent method based on principal amino acid properties. Vaccine. 2007;25:856–66.View ArticlePubMedGoogle Scholar
- Doytchinova IA, Flower DR. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinform. 2007;8:4.View ArticleGoogle Scholar
- Ray P, Sahoo N, Singh B, Kironde FA. Serum antibody immunoglobulin G of mice convalescent from Plasmodium yoelii infection inhibits growth of Plasmodium falciparum in vitro: blood stage antigens of P. falciparum involved in interspecies cross-reactive inhibition of parasite growth. Infect Immun. 1994;62:2354–61.PubMedPubMed CentralGoogle Scholar
- Peng K, Goh YS, Siau A, Franetich JF, Chia WN, Ong AS, et al. Breadth of humoral response and antigenic targets of sporozoite-inhibitory antibodies associated with sterile protection induced by controlled human malaria infection. Cell Microbiol. 2016;18:1739–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Fraser T, Michon P, Barnwell JW, Noe AR, Al-Yaman F, Kaslow DC, et al. Expression and serologic activity of a soluble recombinant Plasmodium vivax Duffy binding protein. Infect Immun. 1997;65:2772–7.PubMedPubMed CentralGoogle Scholar
- Snounou G, Singh B. Nested PCR analysis of Plasmodium parasites. Methods Mol Med. 2002;72:189–203.PubMedGoogle Scholar
- Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988;16:10881–90.View ArticlePubMedPubMed CentralGoogle Scholar
- Bitencourt AR, Vicentin EC, Jimenez MC, Ricci R, Leite JA, Costa FT, et al. Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3. PLoS ONE. 2013;8:e56061.View ArticlePubMedPubMed CentralGoogle Scholar
- Ghai M, Dutta S, Hall T, Freilich D, Ockenhouse CF. Identification, expression, and functional characterization of MAEBL, a sporozoite and asexual blood stage chimeric erythrocyte-binding protein of Plasmodium falciparum. Mol Biochem Parasitol. 2002;123:35–45.View ArticlePubMedGoogle Scholar
- Kappe SH, Gardner MJ, Brown SM, Ross J, Matuschewski K, Ribeiro JM, et al. Exploring the transcriptome of the malaria sporozoite stage. Proc Natl Acad Sci USA. 2001;98:9895–900.View ArticlePubMedPubMed CentralGoogle Scholar
- Doolan DL, Dobaño C, Baird JK. Acquired immunity to malaria. Clin Microbiol Rev. 2009;22:13–36.View ArticlePubMedPubMed CentralGoogle Scholar
- Reyes C, Patarroyo ME, Vargas LE, Rodríguez LE, Patarroyo MA. Functional, structural, and immunological compartmentalisation of malaria invasive proteins. Biochem Biophys Res Commun. 2007;354:363–71.View ArticlePubMedGoogle Scholar
- Xu L, Pei X, Berzins K, Chaudhuri A. Plasmodium yoelii: experimental evidences for the conserved epitopes between mouse and human malaria parasite, Plasmodium falciparum. Exp Parasitol. 2007;116:214–24.View ArticlePubMedGoogle Scholar
- Carlton JM, Vinkenoog R, Waters AP, Walliker D. Gene synteny in species of Plasmodium. Mol Biochem Parasitol. 1998;93:285–94.View ArticlePubMedGoogle Scholar
- Carlton J, Silva J, Hall N. The genome of model malaria parasites, and comparative genomics. Curr Issues Mol Biol. 2005;7:23–37.PubMedGoogle Scholar
- Holder AA, Freeman RR, Newbold CI. Serological cross-reaction between high molecular weight proteins synthesized in blood schizonts of Plasmodium yoelii, Plasmodium chabaudi and Plasmodium falciparum. Mol Biochem Parasitol. 1983;9:191–6.View ArticlePubMedGoogle Scholar
- Taylor DW, Kim KJ, Munoz PA, Evans CB, Asofsky R. Monoclonal antibodies to stage-specific, species-specific, and cross-reactive antigens of the rodent malarial parasite, Plasmodium yoelii. Infect Immun. 1981;32:563–70.PubMedPubMed CentralGoogle Scholar