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bioRxiv · 10.1101/2025.09.02.673669

Design of a novel multi-epitope mRNA vaccine against BtHKU5-CoV-2 using immunoinformatics

zheng, N.·Xu, Y.
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Abstract

Bat HKU5-CoV-2 (BtHKU5-CoV-2), a recently discovered bat-infecting merbecovirus, was found to infect human cell lines by utilizing the human angiotensin-converting enzyme 2 (ACE2) receptor, similar to SARS-CoV-2, which caused millions of deaths. Moreover, its broad host tropism has raised significant concerns about potential human spillover risk. Therefore, there is an urgent need to develop vaccines to combat the potential outbreak of BtHKU5-CoV-2. However, research focusing on BtHKU5-CoV-2 remains limited. In this study, we designed a novel multi-epitope vaccine against BtHKU5-CoV-2 using an immunoinformatic approach. Eight cytotoxic T lymphocyte (CTL) epitopes, seven helper T lymphocyte (HTL) epitopes, and five linear B lymphocyte (LBL) epitopes were screened from the spike glycoprotein of BtHKU5-CoV-2. The selected epitopes were joined together with an appropriate linker, and {beta}-defensin II and MHC I-targeting domain (MITD) were incorporated into the construct to enhance vaccine immunogenicity. Biological characteristic analysis revealed that the designed vaccine exhibited strong antigenicity and immunogenicity while being non-toxic and non-allergenic. The tertiary structure of the multi-epitope vaccine was modeled, refined, and validated, demonstrating its structural stability and near-native conformation. Molecular docking studies showed that the vaccine successfully docked with Toll-like receptor 2 (TLR2) and TLR4. Moreover, its mRNA exhibits strong interactions with TLR3, TLR7, and TLR8 receptors. Additionally, in silico immune simulations have suggested that vaccination could trigger robust humoral and cellular immunity. These findings suggest that the proposed mRNA vaccine is a potential candidate for targeting BtHKU5-CoV-2. Further experiments are necessary to validate its protective efficacy. Author summaryBtHKU5-CoV-2, a newly discovered merbecovirus isolated from bats, exhibits potential for spillover into humans. It was found to utilize human ACE2 as functional receptors for infection. A functional receptor acts like a "key" that fits into the "lock" on the host cell, enabling viral entry. BtHKU5-CoV-2 warrants significant attention, because it shares the same functional receptor with SARS-CoV-1 and SARS-CoV-2, which caused the 2003 SARS epidemic and the 2019 pandemic, respectively. Thus, developing vaccines to prevent potential global outbreaks of BtHKU5-CoV-2 is urgently needed. Theoretically, within the bodys immune surveillance system, proteins from BtHKU5-CoV-2 are processed via proteasomal degradation into short peptides. The peptides with immunogenicity bind to MHC molecules and are presented on the cell surface. These peptides, known as epitopes, can initiate immune reaction. In this study, we designed a multi-epitope mRNA vaccine against BtHKU5-CoV-2 using immunoinformatics methods. Epitopes were screened from the spike glycoprotein, a promising target of BtHKU5-CoV-2. Our results suggest that the vaccine is safe and capable of inducing strong humoral and cellular immunity. Therefore, this mRNA vaccine represents a promising candidate for preventing furture BtHKU5-CoV-2 outbreak.

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zheng, N., Xu, Y.. 2025-09-02. Design of a novel multi-epitope mRNA vaccine against BtHKU5-CoV-2 using immunoinformatics. https://doi.org/10.1101/2025.09.02.673669

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