Designing a Polytopic Protective Antigen Against Echinococcus granulosus for the Both of Definitive and Intermediate Hosts

Introduction: The hydatid disease is one of the most important zoonotic parasitic infections all-round the world. The main part of Echinococcus granulosus life cycle depends upon the growth and differentiation of protoscoleces (PSCs) within the intestine of definitive hosts (Carmena and Cardona 2014). The survival of organism is mainly dependent on its indirect transmission cycle from the definitive hosts (particularly dogs) to the intermediate hosts, including sheep and human. According to the latest updated report of the World Health Organization (WHO) in March 2017, there may be over one million infected cases with echinococcosis at any time (WHO, 2017). A hydatid cyst (HC) encompasses numerous PSCs and cyst fluid, and is formed in visceral organs (liver and lung) of the infected intermediate hosts (Bingham et al. 2014). Morphogenesis of PSCs from the cystic viscera into worm’s head occurs on the surface of the small intestine of the definitive host, and then the head-like structure attaches to the gut epithelial lining and develops into an adult worm within ~50 days (Moro and Schantz 2009). The currently used treatment modalities against echinococcosis, as multi-stage parasitic infection, are based on the anthelmintic drugs such as praziquantel. In this case there is an emergency to improve preventive interventions such as vaccination in addition to hygiene practices. Several constraining factors may influence the vaccine development against such multi-stage pathogens, including economic, socio-cultural issues (Bethony et al. 2011). Thus, it is necessary to implement a rationalized approach towards construction of multipotent vaccines. In silico modeling of vaccines provides a cost- and time-effective approach that can improve such developing effective vaccines (Gori et al. 2013). Ideally, a vaccine construct, to be highly effective, should encompass several parts, including epitopes of one/more VCAs, B-cells epitope and T-cells epitopes (BEs and TEs, respectively) (Pourseif et al. 2017a). Altogether, epitope-based vaccines (EVs) seem to be one of the most effective vaccines. The aim of this study is to design a novel multi-epitope B- and helper T-cell based vaccine construct for immunization of both dog and sheep against this multi-host parasite.
Material and Methods: After antigen sequence selection (GenBank: AEA09024 for eg95 and GenBank: AMX81438 for eg1433), three-dimensional structure of the antigens was modeled and multilaterally validated. The preliminary parameters for B-cell epitope prediction were implemented such as probably transmembrane helix, signal peptide, post-translational modifications. The high ranked B-cell epitopes derived from several online web-servers (e.g., BepiPred v1.0, BcePred, ABCpred, SVMTrip, IEDB algorithms, SEPPA v2.0 and Discotope v2.0) were utilized for multiple sequence alignment and then for engineering the vaccine construct. T-helper based epitopes were predicted by docking between the high frequent Ovar class II allele (Ovar-DRB1*1202) and Dog class II allele (DLA-DRB1*01501) and hexadecamer fragments of the antigens. Having used the immune-informatics tools, we formulated the first bi-valent vaccine based on T-helper epitope with high-binding affinity to sheep and dog MHC alleles. The final vaccine construct was formed by using the molecular spacers and then analyzed for different physicochemical properties.
Results and Discussion:The results of different predictor tools showed that there were four and two potential glycosylation sites in eg1433 and eg95 antigens, respectively. In eg1433 antigen was not observed any transmembrane topology and signal peptide in the protein sequence, however in eg95 antigens was observed a signal peptid residue (aa 1 - 17) and transmembrane fragment (140-MTSGSALTSAIAGFVFSCIVVVL-162). Based on the axiom of immune system properties in response to the more accessible part of the antigens (Ranjbar et al. 2015), in our work we do not considered the transmembrane and signal peptide regions for epitope prediction. The post-translational modification in eg1433 (aa 3, 68, 233, and 238) and eg95 (aa 62 and 70) were also filtered during in silico epitope mapping. These post translationally modified residues are covered by different types of carbohydrate chains and can not likely interact with the immune system elements (Reverberi and Lorenzo 2007). The modeling quality indices (DOPE score and GA431) of the eg95 antigen were -16677.38 kcal/mol and 0.98. The quality scores for eg1433 were -19188.43 kcal/mol and 1.0. These values showed that the structure modeling is implemented with high quality. Some of our predicted epitopes of eg95 antigen were previously reported by Woollard et al. (1998) based on the wet-lab epitope mapping methods (Woollard et al. 1998). However, the eg1433-based epitopes that predicted in our study are for the first time repoted. T-cell epitopes of eg95 and eg1433 (aa 33 – 48 and aa 60 – 72, respectively) were the residues that predicted by docking-based methods. This type of in silico epitope mapping against E. granulosus antigens was not reported previously. The overall processes for establishing such EBVs are as follows: (i) identification and selection of the best antigen from the local and/or global strains, (ii) utilize of bioinformatics tools for in silico analysis of different parameters of selected antigen(s), (iii) computational-based epitope prediction, and (iv) linking epitopes using proper molecular linkers (Toussaint and Kohlbacher 2009).
Conclusion: In this in silico study, we represented a key data on the step-by-step methodologies used for designing this minigene vaccine. It can be as a promising platform for generation of broadly protective host-specific vaccine against E. granulosus.