Immunoinformatic Aided Design of Conserved Peptides Containing Multi-Epitopes against Severe Fever with Thrombocytopenia Syndrome

Abstract

Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) is an emerging zoonotic pathogen, primarily transmitted by Haemaphysalis longicornis ticks, with additional humanto-human and animal-to-human transmission routes. The case fatality rate associated with Severe Fever with Thrombocytopenia Syndrome (SFTS) caused by the virus ranges from 5% to over 30%, varying by demographic and regional factors. Vaccine development remains challenging due to SFTSV's segmented genome, frequent genetic reassortment, and considerable diversity; to date, no licensed vaccine is available. In this study, an immunoinformatics-based strategy was employed to design a multiepitope peptide vaccine targeting the conserved, immunogenic nucleocapsid (N) protein. A total of 1,872 N protein sequences were curated to identify four conserved peptide regions (P1–P4). Advanced machine learning tools (NetMHCpan, MHCflurry, NetMHCIIpan, DeepMHCII, CLBtope) were applied to predict T and B cell epitopes, enabling broad HLA class I (11,576 alleles) and class II (5,625 alleles) coverage. Further antigenicity and allergenicity assessments refined the selection to three peptides (P1–P3). Molecular docking analysis (HPEPDOCK) affirmed robust binding affinity of these peptides (particularly P2 and P3) to a diverse set of HLA molecules (diverse ten alleles for each HLA class). The three selected peptides were linked via flexible spacers to generate six construct combinations. Tertiary structure prediction (Robetta) and subsequent stability evaluations (Robetta confidence, ERRAT, Ramachandran scores) identified four stable constructs (C1, C2, C5, C6). These constructs were further assessed through docking with Tolllike receptor 4 (TLR-4) using the HDOCK server to evaluate innate immune activation potential. Notably, the C5 construct (P3-P1-P2 arrangement) displayed superior and native-like TLR-4 binding efficiency. Collectively, our findings highlight the immunogenic promise of these multi-epitope peptide constructs (especially C5) as valuable candidates for further experimental validation in the pursuit of an SFTSV vaccine.