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 for BtHKU5-CoV-2 using an immunoinformatics 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 appropriate linkers, and β-defensin II and MHC I-targeting domain (MITD) were incorporated into the construct to enhance vaccine immunogenicity. In silico analyses suggested that the designed vaccine may have favorable predicted antigenicity and immunogenicity while being non-toxic and non-allergenic. The tertiary structure of the multi-epitope vaccine was modeled and refined, and its structural plausibility was evaluated using in silico quality metrics. Molecular docking studies suggested plausible interaction modes between the vaccine construct and Toll-like receptor 2 (TLR2) and TLR4. Moreover, the mRNA was predicted to show potential interaction modes with TLR3, TLR7, and TLR8 receptors. Additionally, in silico immune simulations suggested that vaccination may elicit humoral and cellular immune responses. Collectively, these computational results suggest that the proposed mRNA vaccine is a potential candidate for BtHKU5-CoV-2. However, further experiments are necessary to validate its protective efficacy.
