Skip to main content

Epitope vaccine design for Toxoplasma gondii based on a genome-wide database of membrane proteins



There is presently no effective and safe vaccine for Toxoplasma gondii for humans. The study described here was designed to search for a novel group of optimal B cell and T cell epitopes from Toxoplasma membrane proteins using genome-wide comprehensive screening.


The amino acid sequences of membrane proteins of T. gondii were obtained from the UniProt database. The ABCPred and BepiPred servers were employed to predict the linear B cell epitopes. The Immune Epitope Database (IEDB) online service was utilized to forecast T cell epitopes within T. gondii membrane proteins that bind to human leukocyte antigen (HLA) class I (HLA-I) or HLA-II molecules.


From the 314 membrane proteins of T. gondii, a total of 14 linear B cell epitopes embedded in 12 membrane proteins were identified. Eight epitopes for major histocompatibility complex (MHC) class I (MHC-I) molecules and 18 epitopes for MHC-II molecules were ultimately selected, for which world population coverage percentiles were 71.94% and 99.76%, respectively. The top rated combinations of linear B cell epitopes and T cell epitopes covering both BALB/c mice and a majority of the human population were identified for the development of a protective vaccine.


The ultimate vaccine construct described here, which comprises B cells, MHC-I and MHC-II epitopes, might protect individuals against T. gondii infection by inducing humoral and cellular immune responses.

Graphic abstract


Toxoplasma gondii is an intracellular parasite that causes toxoplasmosis in humans and animals [1, 2]. Infection with this parasite during pregnancy can lead to infection of the fetus, and may cause miscarriage or stillbirth [3]. Moreover, this infection can be lethal in immunocompromised individuals. Toxoplasmosis is also prevalent among domestic animals, and may result in abortion, especially in goats and sheep, which can cause enormous losses to the livestock industry [4]. Thus, protecting humans and livestock from Toxoplasma infection is important for public health.

Vaccination is a more effective approach for T. gondii than chemotherapy [5]. However, there is as yet no effective and safe vaccine for T. gondii for humans, mainly due to limitations in vaccine development strategies and lack of good candidate vaccine molecules [5]. Most of the current studies on epitopes only focus on some characterized proteins of T. gondii, and there has been no extensive and systematic search of Toxoplasma proteins. Moreover, experimental results derived from mice, the most widely used animal model for T. gondii vaccine development, are inevitably biologically biased due to the genetic differences between mice and humans [6].

The present study was designed to unbiasedly search for a novel group of optimal B cell and T cell epitopes. Bioinformatics and immunoinformatic analysis were conducted for a genome-wide range of Toxoplasma membrane proteins (including uncharacterized proteins). The steps of the in silico analysis are illustrated in Fig. 1.

Fig. 1
figure 1

Workflow diagram


Linear B cell epitope prediction

Surface proteins and cell membrane proteins were identified as targets. The amino acid sequences of membrane proteins of T. gondii were obtained from the UniProt database ( ABCPred [7], and the BepiPred [8] servers were employed to predict the linear B cell epitopes. The window length of the ABCPred server prediction results was set to 16, and the threshold value adjusted to 0.95 due to the large number of predictions obtained. For BepiPred server 2.0, the threshold value was set to 1.5 to screen for epitopes with high scores. When the results of the ABCPred and BepiPred servers converged, we used the combined results as the candidate linear B cell epitopes for the subsequent analysis.

Prediction of mutual epitopes of human leukocyte antigens and mouse major histocompatibility complex

The Immune Epitope Database (IEDB) online service was utilized to forecast T cell epitopes within T. gondii membrane proteins that bind to human leukocyte antigen (HLA) class I (HLA-I) or HLA-II molecules [9, 10]. For HLA-I molecules, the HLA allele reference set was selected and peptides with assessment scores greater than 0.7 were selected in the screening. For HLA-II molecules, the full HLA reference set was selected and the peptides in the top 0.5% were selected as the screening range for the prediction results. Computation of the combined population coverage was conducted using the Population Coverage tool [11].

BALB/c mice, the laboratory animals most commonly used in vaccine studies, were selected as the targets to screen for major histocompatibility complex (MHC) molecules that bind to epitopes. Three MHC class I (MHC-I) alleles (H2-Db, H2-Kb and H2-Ld) and two MHC-II alleles (H2-IAd and H2-IEd) from BALB/c mice were employed [9, 10, 12]. We retained the MHC-I epitopes with scores greater than 0.7 and the MHC-II epitopes that were ranked in the top 0.5%. The epitopes’ binding abilities for each allele were evaluated and listed.

Profiling and evaluation of predicted epitopes

Vaxijen2.0 server [13], AllerTOP v. 2.0 [14] and ToxinPred [15] were employed to evaluate the antigenicity, allergenicity and toxicity of all epitopes; these tools were applied using their default settings. The SignalP 5.0 server was used [16] to exclude the epitopes which embody signal peptides. The TMHMM 2.0 server [17] was utilized to analyze the transmembrane situation of epitope-derived proteins. Epitopes located in the outer membrane at a probability greater than 0.85 were recruited. ToxinPred was employed to screen for epitopes with good hydrophilicity.

Results and discussion

Over a third of the global human population is in danger of T. gondii infection. The currently available anti-toxoplasmosis vaccines, including live attenuated vaccines, lysis products and excreted/secreted antigen vaccines, are all for non-human use [18]. Live attenuated vaccines emulated natural infection and provided a microenvironment for antigen processing and presentation similar to that of T. gondii infection [18,19,20]. A commercial vaccine, Toxovax, based on the live attenuated tachyzoites of the S48 strain of T. gondii, has been licensed for use in New Zealand, the UK and some European countries to reduce the incidence of abortion in sheep that results from infection [21]. Most of the current anti-toxoplasmosis vaccines were developed by targeting a number of protein families such as surface antigens, rhoptry bulb proteins, microneme proteins, and dense granule proteins as antigens [18,19,20, 22].

Currently, antigen selection is mostly based on proteins identified without an extensive search of the Toxoplasma gene database. Large-scale screening would enhance the likelihood of identifying potentially promising epitopes. In order to select the best epitopes, we targeted membrane proteins of T. gondii from the UniProt database, and obtained 314 of them. Proteins without an extracellular domain were excluded from the following. Information on the proteins is presented in Additional file 1 and their amino acid sequences in Additional file 2. A total of 238 epitopes were predicted by the ABCPred server with the threshold value set to 0.95 (Additional file 3). Of these predicted epitopes, 32 peptides were also identified by BepiPred 2.0 server (Additional file 15: Table S1). Fourteen linear B cell epitopes with high scores of antigenicity computed via vaxijen2.0 were selected. These 14 B cell epitopes of T. gondii were distributed on 12 membrane proteins (Table 1); their immunobiological characteristics are presented in Additional file 16: Table S2. The overlapping parts of the epitopes identified by both the ABCPred and BepiPred-2.0 servers are shown in Additional file 11: Figure S1. The distribution of these epitopes on the corresponding proteins is shown in Additional file 12: Figure S2. All of these epitopes have probabilities > 0.85 of being exposed towards the outside of cells.

To the best of our knowledge, this work is the first systematic epitope vaccine screening of all T. gondii membrane proteins through bioinformatics analysis. Within this wide range of target proteins, the retained B cell and T cell epitopes were non-allergenic, non-toxic, and had good antigenicity. For B cell epitopes, we also proceeded with the prediction of signal peptides, hydrophilicity and transmembrane motifs in the proteins.

Table 1 Summary of the selected linear B cell epitopes and corresponding proteins

The population coverage of the HLA alleles was predicted by the Population Coverage tool of IEDB Analysis Resource ( The global population coverage rate as well as those of 11 major regions of the world were computed. For HLA-I, nine world regions showed more than 50% population coverage, with the highest population coverage in Europe, at 81.48%, and in North America, at 76.79% (Additional file 13: Figure S3a). For HLA-II, the population coverage rates of all 11 world regions exceeded 95% (Additional file 13: Figure S3b). T cell epitope screening of the 314 membrane proteins of T. gondii was conducted using the IEDB online service. For HLA-I binding epitopes, the selected scores ranged from > 0.7, and we obtained 1221 initial predictions (Additional file 4). For HLA-II binding epitopes, results with prediction scores that were ranked in the top 0.5% were selected, which gave 19,241 initial epitopes candidates (Additional file 5).

Because of the inherent genetic differences between mice and humans, conclusions based on the results of experiments using mice cannot be directly extrapolated to humans. For example, immunity-related guanosine triphosphatases assist mice during early Toxoplasma infection, but humans lack most of the genes in this protein family [23]. Nevertheless, in vivo experimental validation using mice is still valuable and important. In sum, the designed epitopes should ideally be recognized by both HLA and mouse MHC. The T-cell epitope prediction using mouse MHC molecules was conducted online, and resulted in 920 mouse MHC-I binding epitopes (H2-Db, H2-Kb and H2-Ld) with a score > 0.7 (Additional file 6) and 1185 mouse MHC-II binding epitopes (H2-IAd, H2-IEd] that were ranked in the top 0.5% (Additional file 7). Subsequently, we identified 909 epitopes capable of binding to both HLA-I and mouse MHC-I molecules, and 526 epitopes capable of binding to both HLA-II and mouse MHC-II molecules (Additional file 8). The epitopes with the highest score or highest rank were selected. After screening for antigenicity, allergenicity and toxicity, eight MHC-I binding epitopes were retained; these could be recognized by 12 HLA-I alleles and three mouse MHC-I alleles (Table 2). For MHC-II binding peptides, we selected 18 epitopes that can bind to 19 HLA-II alleles and two mouse MHC-II alleles (Table 2). The alleles covered and the corresponding scores or ranks of each epitope are shown in Additional file 14: Figure S4. As the selected epitopes could be recognized by both human and mouse MHC molecules, our strategy may be a successful approach to translating mouse-derived experimental results to humans for future vaccine development.

Table 2 Human leukocyte antigen (HLA) I and HLA II allele-binding epitopes predicted by the Immune Epitope Database server

The tentative vaccine design contains three linear B cell epitopes, three MHC-I epitopes and two MHC-II epitopes (Fig. 2a). The B cell epitopes were chosen according to a holistic consideration of their antigenicity and hydrophilicity. For T cell epitopes, the selection was based on the corresponding score/rank and the population coverage. The identified epitopes all exhibit strong binding affinity to a series of MHC molecules (Additional file 9). The selected T cell epitopes could theoretically be recognized and presented by immune cells in more than 96% of the world’s population (Additional file 10). In addition, full coverage for three MHC-I and two MHC-II alleles in BALB/c mice indicates that there will also be a strong immune response in laboratory mice. The epitopes determined in this study were linked using (GGGGS)2 linkers for a chimeric antigen (Fig. 2b).

Fig. 2a, b
figure 2

Epitope-based vaccine construct. a Schematic view of the vaccine design. b Amino acid sequence of the vaccine construct.

Despite the findings discussed above, the current study had several limitations. Firstly, the epitopes predicted and selected in this study need to be validated by in vitro or in vivo experiments. Secondly, both the efficacy and safety of the construct as an anti-Toxoplasma vaccine need to be tested. Thirdly, the current study focused on the epitope screening of Toxoplasma membrane proteins, but good epitope candidates may also exist that are located on cytosolic proteins.

Some of the epitopes identified here matched those from previous studies [18,19,20, 24]. In addition, some of the proteins from which they were identified are putative membrane proteins, and the T and B cell epitopes screened using them also exhibited excellent immunogenicity. Thus, these are potentially excellent candidate molecules. The search of the IEDB server showed that the (S8F3F6)990–1008 and (AOA125YINI)480–494 peptides in the vaccine construct, which are experimentally determined epitopes (Additional file 17: Table S3), harbor homologous sequences to yellow fever virus and Yersinia pestis, respectively. Admittedly, although in silico analysis is a powerful forecasting tool, its results are not substitutes for experimental evidence. Thus, the immunological characteristics of the epitopes identified here will be further evaluated in both in vitro and in vivo experiments.

Availability of data and materials

Not applicable.



Human leukocyte antigen


Immune Epitope Database


Major histocompatibility complex


  1. Dubey JP. The history of Toxoplasma gondii–the first 100 years. J Eukaryot Microbiol. 2008;55:467–75.

    Article  Google Scholar 

  2. Rostami A, Riahi SM, Fakhri Y, Saber V, Hanifehpour H, Valizadeh S, et al. The global seroprevalence of Toxoplasma gondii among wild boars: a systematic review and meta-analysis. Vet Parasitol. 2017;244:12–20.

    Article  Google Scholar 

  3. Fallahi S, Rostami A, Nourollahpour Shiadeh M, Behniafar H, Paktinat S. An updated literature review on maternal-fetal and reproductive disorders of Toxoplasma gondii infection. J Gynecol Obstet Hum Reprod. 2018;47:133–40.

    Article  CAS  Google Scholar 

  4. Foroutan M, Rostami A, Majidiani H, Riahi SM, Khazaei S, Badri M, et al. A systematic review and meta-analysis of the prevalence of toxoplasmosis in hemodialysis patients in Iran. Epidemiol Health. 2018;40:e2018016.

    Article  Google Scholar 

  5. Wang JL, Zhang NZ, Li TT, He JJ, Elsheikha HM, Zhu XQ. Advances in the development of Anti-Toxoplasma gondii vaccines: challenges, opportunities, and perspectives. Trends Parasitol. 2019;35:239–53.

    Article  CAS  Google Scholar 

  6. Szabo EK, Finney CAM. Toxoplasma gondii: one organism, multiple models. Trends Parasitol. 2017;33:113–27.

    Article  Google Scholar 

  7. Saha S, Raghava GP. Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins. 2006;65:40–8.

    Article  CAS  Google Scholar 

  8. Jespersen MC, Peters B, Nielsen M, Marcatili P. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes. Nucleic Acids Res. 2017;45:W24–9.

    Article  CAS  Google Scholar 

  9. Andreatta M, Nielsen M. Gapped sequence alignment using artificial neural networks: application to the MHC class I system. Bioinformatics. 2016;32:511–7.

    Article  CAS  Google Scholar 

  10. Nielsen M, Lundegaard C, Lund O. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method. BMC Bioinformatics. 2007;8:238.

    Article  Google Scholar 

  11. Bui HH, Sidney J, Dinh K, Southwood S, Newman MJ, Sette A. Predicting population coverage of T-cell epitope-based diagnostics and vaccines. BMC Bioinformatics. 2006;7:153.

    Article  Google Scholar 

  12. Zhang GL, Srinivasan KN, Veeramani A, August JT, Brusic V. PREDBALB/c: a system for the prediction of peptide binding to H2d molecules, a haplotype of the BALB/c mouse. Nucleic Acids Res. 2005;33:W180-3.

    Article  CAS  Google Scholar 

  13. Doytchinova IA, Flower DR. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics. 2007;8:4.

    Article  Google Scholar 

  14. Dimitrov I, Bangov I, Flower DR, Doytchinova I. AllerTOP vol 2–a server for in silico prediction of allergens. J Mol Model. 2014;20:2278.

    Article  Google Scholar 

  15. Gupta S, Kapoor P, Chaudhary K, Gautam A, Kumar R, Raghava GP. Peptide toxicity prediction. Methods Mol Biol. 2015;1268:143–57.

    Article  CAS  Google Scholar 

  16. Almagro Armenteros JJ, Tsirigos KD, Sonderby CK, Petersen TN, Winther O, Brunak S, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol. 2019;37:420–3.

    Article  CAS  Google Scholar 

  17. Moller S, Croning MD, Apweiler R. Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics. 2001;17:646–53.

    Article  CAS  Google Scholar 

  18. Li Y, Zhou H. Moving towards improved vaccines for Toxoplasma gondii. Expert Opin Biol Ther. 2018;18:273–80.

    Article  Google Scholar 

  19. Zhang NZ, Wang M, Xu Y, Petersen E, Zhu XQ. Recent advances in developing vaccines against Toxoplasma gondii: an update. Expert Rev Vaccines. 2015;14:1609–21.

    Article  CAS  Google Scholar 

  20. Hiszczynska-Sawicka E, Gatkowska JM, Grzybowski MM, Dlugonska H. Veterinary vaccines against toxoplasmosis. Parasitology. 2014;141:1365–78.

    Article  Google Scholar 

  21. Buxton D, Innes EA. A commercial vaccine for ovine toxoplasmosis. Parasitology. 1995;110:S11–6.

    Article  Google Scholar 

  22. Chen J, Zhou DH, Li ZY, Petersen E, Huang SY, Song HQ, et al. Toxoplasma gondii: protective immunity induced by rhoptry protein 9 (TgROP9) against acute toxoplasmosis. Exp Parasitol. 2014;139:42–8.

    Article  CAS  Google Scholar 

  23. Howard JC, Hunn JP, Steinfeldt T. The IRG protein-based resistance mechanism in mice and its relation to virulence in Toxoplasma gondii. Curr Opin Microbiol. 2011;14:414–21.

    Article  CAS  Google Scholar 

  24. Torres-Morales E, Taborda L, Cardona N, De-la-Torre A, Sepulveda-Arias JC, Patarroyo MA, et al. Th1 and Th2 immune response to P30 and ROP18 peptides in human toxoplasmosis. Med Microbiol Immunol. 2014;203:315–22.

    Article  CAS  Google Scholar 

Download references


We appreciate the generous technical support of Dr. Ning Zhang. Thanks also to Dr. Modeste Judes and Ms. Shasha Song for proofreading the manuscript and correcting grammatical mistakes.


This work was supported by a National Key Research and Development Project (2018YFE0114500), the National Natural Science Foundation of China (82072306, 81970746, 31571244), and Hunan Provincial Natural Sciences Foundation (2022JJ30797).

Author information

Authors and Affiliations



HZC and XW conceived and designed the project. XWL and NZ performed the bioinformatics analysis. DN undertook the language correction. ZRM, BL, ZLL and YHH helped XWL and NZ analyze the data. XWL, HZC and XW wrote the manuscript. All the authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Hong-Zhi Chen or Xiang Wu.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Consent has been given for publication of all the data presented here.

Competing interests

The authors declare that they have no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1.

Information on membrane proteins of Toxoplasma gondii obtained from the UniProt database.

Additional file 2.

Amino acid sequences of the proteins presented in Additional file 1.

Additional file 3

. Linear B cell epitopes predicted by the ABCPred server.

Additional file 4.

Initial predicted HLA-I binding epitopes.

Additional file 5.

Initial predicted HLA-II binding epitopes.

Additional file 6.

Initial predicted mouse MHC-I binding epitopes.

Additional file 7

. Initial predicted mouse MHC-II binding epitopes.

Additional file 8.

Identified epitopes capable of binding to both HLA-I and mouse MHC-I molecules, and epitopes capable of binding to both HLA-II and mouse MHC-II molecules.

Additional file 9.

Binding capacity of selected T cell epitopes.

Additional file 10.

Worldwide population coverage of HLA alleles by the vaccine.

Additional file 11: Figure S1.

Selected linear B cell epitopes and their positions on the corresponding proteins.

Additional file 12: Figure S2.

Transmembrane motif analysis of proteins pertaining selected linear B cell epitopes. (Arrows and numbers indicate the positions of the epitopes in the protein sequences).

Additional file 13: Figure S3.

Population coverage analysis. (a) Population coverage analysis of the 13 Recommended HLA-I alleles; (b) Population coverage analysis of the 19 Recommended HLA-II alleles

Additional file 14

: Figure S4. Selected T cell binding epitopes. (a) CD8+ T cell epitopes and their corresponding MHC-I alleles in mice and human; (b) CD4+ T cell epitopes and their corresponding MHC-II alleles in mice and human

Additional file 15: Table S1.

The initial result of the joint prediction using BepiPred-2.0 and ABCPred.

Additional file 16: Table S2

Antigenicity, allergenicity, toxicity, transmembrane localization, signal peptide and hydrophilicity profiling of selected peptides.

Additional file 17: Table S3

Alignment of experimentally verified epitopes and selected sequences in the vaccine construct.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, XW., Zhang, N., Li, ZL. et al. Epitope vaccine design for Toxoplasma gondii based on a genome-wide database of membrane proteins. Parasites Vectors 15, 364 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Toxoplasma gondii
  • Epitope vaccine
  • Bioinformatics
  • Human leukocyte antigen