همایش ملی بیوانفورماتیک ایران

صفحه اصلی / 4th international edition and 13th Iranian Conference on Bioinformatics

Epitope-Based Design of a Dual-Purpose Recombinant Protein Targeting Dengue NS1 for Vaccine and Diagnostic Development

نویسندگان :

Abolhassan Bahari¹ Amirmahdi Yavari²

1- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran 2- Department of Biology, Faculty of Sciences, Central Tehran Branch, Islamic Azad University, Tehran, Iran

کلمات کلیدی :

Dengue virus،vaccine design،immunoinformatics،NS1 protein،recombinant protein

چکیده :

Dengue fever is a mosquito-borne viral disease caused by four dengue virus serotypes of the Flaviviridae family. It is a major global health concern, with a wide spectrum of symptoms, ranging from mild febrile illness to severe forms like dengue hemorrhagic fever and dengue shock syndrome (Kularatne and Dalugama, 2022). Among the components of the dengue virus, the nonstructural protein 1 (NS1), secreted during infection, plays a pivotal role in immune evasion and pathogenesis. NS1 is a highly immunogenic protein, making it an ideal target for both diagnostic and vaccine development (Munasinghe et al., 2022). This study aims to design a dual-purpose recombinant protein, serving as both an ELISA kit component and a vaccine candidate, by identifying NS1-derived epitopes through in silico analysis. Since there is an overlap in the design principles of vaccines and diagnostic kits, this study emphasizes the selection of epitopes that are conserved (across DENV serotypes), immunogenic, non-pathogenic, non-toxic, safe, non-allergenic, and of wide applicability in the Iranian population. The NS1 protein was first screened using BLASTp to confirm no cross-reactivity with human proteins (Altschul et al., 1997). No similar eukaryotic proteins found. Membrane topology of NS1 protein regions predicted using DeepTMHMM to identify secretable regions (Hallgren et al., 2022). Immunogenicity was assessed with VaxiJen 3.0 (Doneva and Dimitrov, 2024). conserved regions were identified using EMBL-EBI ClustalW (Madeira et al., 2024). T-cell epitopes for MHC I and MHC II were predicted using IEDB tools (Reynisson et al., 2020, Nilsson et al., 2023). For MHC I epitopes, criteria included percentile rank (<0.5) and repeat frequency (>4), yielding 40 epitopes with 97.6% population coverage in Iran. For MHC II, epitopes with similar criteria achieved 97% population coverage (Bui et al., 2006, Abedini et al., 2021). Linear and non-linear B-cell epitopes were predicted using BepiPred3, DiscoTope-3.0 and ElliPro (Clifford et al., 2022, Høie et al., 2024, Ponomarenko et al., 2008) and filtered by length ≥ 10 amino acids and immunogenicity scores. Further, the MHC II epitopes were analyzed for their induction capabilities of IFN-gamma, IL4, and IL10 by using IFNepitope, IL4 Pred, and IL10 Pred (Dhanda et al., 2013b, Dhanda et al., 2013a, Nagpal et al., 2017). Epitopes inducing all three cytokines were selected, totaling four. We also searched literature to find experimentally and clinically validated epitopes. Toxicity, allergenicity and antigenicity validations for 23 experimental and 19 predicted epitopes using ToxinPred3.0, AllerTop2.1 and VaxiJen3.0 online webservers ensured the safety of selected epitopes (Dimitrov et al., 2014, Rathore et al., 2024). Conservancy of epitopes across four DENV serotypes measured by IEDB tools (Bui et al., 2007). The final recombinant protein included five computationally predicted and seven experimentally and computationally validated epitopes, linked with standard linker peptides and HisTag for recombinant protein expression and purification. This work presents a tight epitope selection process for ensuring immunogenicity and safety, supporting broad Iranian population coverage for both vaccine and diagnostic kits. The integrated approach addresses global health challenges presented by dengue and thus opens up further structural and experimental validation.