طراحی آنتی ژن حفاظتی پلی توپیک علیه انگل اکینوکوکوس گرانولوزوس برای هر دو میزبان اصلی و واسط

زمینه مطالعاتی: انگل اکینوکوکوس گرانولوزوس عامل بیماری زئونوتیک کیست هیداتیک است که در بسیاری از واحدهای دامپروری و بویژه در مناطق روستایی و حومه شهرها به لحاظ اپیدمیولوژی در وضعیت اندمیک است. میزبان‌های طبیعی این پاتوژن انگلی به طور عمده سگ (به عنوان میزبان اصلی)، گوسفند و انسان (به عنوان میزبان واسط) هستند. امروزه کنترل و پیشگیری این بیماری عمدتا محدود به اجرای برنامه‌های آموزشی، نظارتی و مدیریتی در مزارع دامپروری و کشتارگاه‌ها بوده است. در دهه اخیر توجه جدی به توسعه واکسن پیشگیرانه بوجود آمده است. هدف از این مطالعه معرفی ابزارها و نرم افزارهای بیوانفورماتیک هستند که در ‌طراحی آنتی ژن حفاظتی مفید خواهند بود. روش کار: در این مطالعه از ابزارهای پیشگویی کننده ی اپی‌توپ مربوط به مربوط به سیستم ایمنی سلولی و هومورال استفاده گردید. همچنین از نرم افزار مدلر برای مدلینگ ساختار سوم هر یک از آنتی ژن های حفاظتی استفاده گردید. داکینگ مولکولی بین مولکول های mhc اختصاصی گونه و نیز پپتیدهای کاندید اپی توپ سلول t مورد استفاده قرار گرفت. برای این کار از نرم افزار hex ورژن 8 استفاده شد. نتایج: شاخص های ایمونوژنیک مربوط به هر یک از آنتی‌ژن ها در قالب یک سازه آنتی ژن حفاظتی مرتب گردید و توسط لینکرهای اختصاصی در کنار هم قرار گرفتند. سپس کدون ها ی این سازه آنتی ژن حفاظتی برای بیان در میزبان پروکاریوتی (escherichia coli k12) بهینه گردید. نتیجه گیری نهایی: در این مطالعه ی بیوانفورماتیک، اطلاعات کلیدی در خصوص روش شناسی گام به گام طراحی دنوو آنتی ژن حفاظتی علیه بیماری انگلی اکینوکوکوس گرانولوزوس ارائه شده است. که به نظر می رسد قابلیت حفاظت همزمان سگ و گوسفند در برابر این بیماری را داشته باشد.

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 allround 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 headlike 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 multistage 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 multistage pathogens, including economic, sociocultural 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 timeeffective 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, Bcells epitope and Tcells epitopes (BEs and TEs, respectively) (Pourseif et al. 2017a). Altogether, epitopebased vaccines (EVs) seem to be one of the most effective vaccines. The aim of this study is to design a novel multiepitope B and helper Tcell based vaccine construct for immunization of both dog and sheep against this multihost parasite. Material and Methods: After antigen sequence selection (GenBank: AEA09024 for eg95 and GenBank: AMX81438 for eg1433), threedimensional structure of the antigens was modeled and multilaterally validated. The preliminary parameters for Bcell epitope prediction were implemented such as probably transmembrane helix, signal peptide, posttranslational modifications. The high ranked Bcell epitopes derived from several online webservers (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. Thelper based epitopes were predicted by docking between the high frequent Ovar class II allele (OvarDRB1*1202) and Dog class II allele (DLADRB1*01501) and hexadecamer fragments of the antigens. Having used the immuneinformatics tools, we formulated the first bivalent vaccine based on Thelper epitope with highbinding 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 (140MTSGSALTSAIAGFVFSCIVVVL162). 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 posttranslational 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 wetlab epitope mapping methods (Woollard et al. 1998). However, the eg1433based epitopes that predicted in our study are for the first time repoted. Tcell epitopes of eg95 and eg1433 (aa 33 – 48 and aa 60 – 72, respectively) were the residues that predicted by dockingbased 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) computationalbased 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 stepbystep methodologies used for designing this minigene vaccine. It can be as a promising platform for generation of broadly protective hostspecific vaccine against E. granulosus.