Citation: Dar, H.A.; Almajhdi, F.N.; Aziz, S.; Waheed, Y. Immunoinformatics-Aided Analysis of RSV Fusion and Attachment Glycoproteins to Design a Potent Multi-Epitope Vaccine. Vaccines 2022, 10, 1381. https://doi.org/10.3390/ vaccines10091381 Academic Editor: Toshi Nagata Received: 4 July 2022 Accepted: 20 August 2022 Published: 24 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article Immunoinformatics-Aided Analysis of RSV Fusion and Attachment Glycoproteins to Design a Potent Multi-Epitope Vaccine Hamza Arshad Dar 1 , Fahad Nasser Almajhdi 2 , Shahkaar Aziz 3 and Yasir Waheed 4, * 1 Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea 2 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia 3 Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar 25000, Pakistan 4 Office of Research, Innovation & Commercialization, Shaheed Zulfiqar Ali Bhutto Medical University (SZABMU), Islamabad 44000, Pakistan * Correspondence: director.oric@szabmu.edu.pk or yasir_waheed_199@hotmail.com; Tel.: +92-300-5338171 Abstract: Respiratory syncytial virus (RSV) usually causes respiratory tract infections of upper airways in infants and young children. Despite recent medical advances, no approved vaccine is available to control RSV infections. Therefore, we conducted an immunoinformatics study to design and evaluate a potential multi-epitope vaccine against RSV. Sequence-based analyses of the glyco- proteins F and G revealed a total of eight CD8 T-cell and three CD4 T-cell epitopes after considering antigenicity, binding affinity and other parameters. Molecular docking analysis confirmed that these T-cell epitopes developed strong structural associations with HLA allele(s). By integrating these prioritized epitopes with linkers and a cholera toxin-derived adjuvant, a multi-epitope vaccine was designed. The developed vaccine was found to be stable, non-allergenic, flexible and antigenic. Molecular docking analysis revealed a striking mean HADDOCK score (−143.3) of top-ranked vaccine-TLR cluster and a Gibbs free energy change (ΔG) value of −11.3 kcal mol −1 . As per computa- tional immune simulation results, the vaccine generated a high titer of antibodies (especially IgM) and effector T-cells. Also, codon optimization and in silico cloning ensured the increased expression of vaccine in Escherichia coli. Altogether, we anticipate that the multi-epitope vaccine reported in this study will stimulate humoral and cellular responses against RSV infection, subject to follow-up experimental validation. Keywords: respiratory syncytial virus; multi-epitope vaccine; molecular modeling; molecular dynamics; vaccine candidate 1. Introduction Human respiratory syncytial virus (RSV) belongs to the Pneumoviridae virus family [1,2]. The genome size of this species is ~15.19 kb, and it contains a single-stranded RNA with a negative sense [3,4]. A total of two subtypes of RSV have been reportedly iden- tified, i.e., RSV-A and RSV-B [5,6]. RSV exhibits unique characteristics that distinguish it from its family members. The RSV virion is formed when the viral nucleocapsid is enclosed by a lipid envelope, that in turn is generated from the host cell’s plasma membrane [7]. The RSV genome contains a total of ten genes, including those which encode fusion protein F and attachment glycoprotein G [7,8]. Using the F protein, the virus exhibits binding to the host cell surface [9]. After this successful interaction, RSV enters the host cells and forms “syncytia”. The virus surface-associated fusion and glycoproteins essentially drive the synthesis of neutralizing antibodies in hosts, thus confirming their involvement in pathogenesis [7]. Vaccines 2022, 10, 1381. https://doi.org/10.3390/vaccines10091381 https://www.mdpi.com/journal/vaccines