Design of a Peptide-Based Vaccine Against Human Respiratory Syncytial Virus (hRSV) Using a Reverse Vaccinology Approach: Evaluation of Immunogenicity, Antigenicity, Allergenicity, and Toxicity

Hadeel Alnajran ¹ Dr. Maaweya Awadalla ² Fahad Aldakheel ¹ Intikhab Alam ³ Afaque A Momin ³ Wael Alturaiki ⁴ Bandar Alosaimi ^2*

• ¹ Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia
• ² King Fahd Medical City, Riyadh, Saudi Arabia
• ³ King Abdullah University of Science and Technology, Thuwal, Makkah, Saudi Arabia
• ⁴ College of Applied Medical Sciences, Majmaah University, Majmaah, Saudi Arabia

Background: Attempts to develop an hRSV vaccine have faced safety and efficacy challenges, with only three FDA-approved vaccines (Moderna’s Mresvia, Pfizer’s Abrysvo, and GSK’s Arexvy) available. These vaccines are limited to individuals over 60 years, require boosters, and only reduce disease severity without clearing the infection. Therefore, we employed a reverse vaccinology approach in this study to identify the most promising antigenic epitopes capable of eliciting a robust and protective immune response.Methodology: This study employed computational techniques to design a novel multi-epitope vaccine targeting hRSV. Using bioinformatics tools, candidate epitopes were identified from conserved viral proteins (F and G glycoproteins), assessing their immunogenicity, antigenicity, and allergenicity. Key tools included ExPASy, ProtParam, VaxiJen v2.0, AllergenFP v1.0, AllerTOP v2.0, NetCTL v1.2, IEDB, and Toxin-Pred. The vaccine construct was assessed for stability and toxicity through in silico analyses. We then characterized its kinetic properties, evaluated its structural integrity, and analyzed its interactions with Toll-like receptors (TLRs) using molecular docking, modeling, and refinement with AlphaFold3 and ClusPro. Results: The designed constructs showed strong antigenicity (0.5996 for F-based and 0.6048 for G-based vaccine), non-allergenicity, and stability (instability index <40). Among these, most amino acids were in the extracellular domain of the construct. Molecular docking and dynamics simulations indicated strong binding interactions with TLR1 and TLR4 and minimal RMSF fluctuations, which ensured structural stability. Strong humoral and cellular responses were suggested by in silico immune simulation demonstrating robust immune activation, with high levels of IgG, IgM, IL-2, and IFN-γ. The physical and chemical analyses revealed that the majority of amino acids from the F and G proteins were located in the extracellular domain of the construct. The presence of signal peptide cleavage sites in both glycoprotein components further facilitates antigen presentation to the immune system. Conclusions: This study presents a promising peptide-based vaccine candidate against hRSV that can effectively engage the immune system, showing strong immunogenicity and antigenicity. Future in vitro and in vivo studies are essential to evaluate the ability of the multi-epitope vaccine candidate to stimulate both humoral and cell-mediated immune responses and to assess its efficacy and safety profile.