Design and Validation of a Multi-Epitope mRNA Vaccine Construct Against Human Monkeypox Virus (hMPXV) by Annotating Protein of Intracellular Mature Virus (IMV) Form of hMPXV

<b>Background</b>: hMPXV poses a major public health risk due to its human-to-human transmissibility, severe complications, especially in immunocompromised individuals, and global spread, necessitating effective surveillance and stringent prophylactic measures to mitigate its colossal impact. <b>Objective</b>: The study aimed to annotate hMPXV(IMV) proteins to propose a potential reverse vaccinology-based vaccine against hMPXV. <b>Methods</b>: The target MPXV(IMV) protein's sequences, formatted in FASTA, were sourced from genome/proteome databases (BV-BRC and UniProt) (accessed on 6 November 2024), followed by CD-Hit-based redundancy removal. Epitope prediction for B-cells (lymphocytes), cytotoxic T-cells or cytotoxic T-lymphocytes (CTLs), and helper T-cells (HTLs) was executed using ABCpred, IEDB's ANNs 4.0, and an artificial neural network-based alignment tool (NN-align 2.3)/ML-based tool (NetMHCII 2.3). Various immunoinformatics filters (antigenicity, toxicity, and allergenicity) were applied to substantiate the potency and safety of the formulated vaccine candidate. The constructed vaccine's physiochemical and structural features (secondary and tertiary), with structural stability (confirmed by molecular docking followed by dynamic simulation with TLRs (TLR4 & TLR2) and MHCs), were determined. Additionally, cloning (using pET-28a(+) vector) was conducted to verify the vaccine's expression potential and translation efficiency. The construct's population coverage was also ascertained. <b>Results</b>: The MPXV-2-Beta vaccine constructs, of the six initially designed constructs, was identified as the most promising candidate, signifying nonallergenic profile and nontoxic features, with a predicted antigenicity score (PAS) = 0.7202, 407 residues, a molecular weight of 43,102.1 Da, pI of 9.2, and favorable stability parameters (AI: 65.65, GRAVY: -0.597, I-i: 25.92). It showed high solubility (score: 0.942). The ProSA Z-score of -9.38 confirmed the structural stability, reliability, and precision of the MPXV-2-Beta 3D model, which is comparable to experimental structures. Furthermore, 98.8% of all the residues nested within favored or allowed regions in a critical Ramachandran plot signified the model's exceptional structural integrity and quality. Docking and dynamic simulation of MPXV-2-Beta with TLRs (TLR4 & TLR2) and MHCs demonstrated stiffer docking stability (strong polar and nonpolar interaction) and negative eigenvalue value (during dynamic simulation), suggesting its ability to enhance immune receptor activation under physiological conditions. MPXV-2-Beta was predicted to trigger a robust immune response (IR) with comprehensive world population coverage (98.55%, SD = 10.41). <b>Conclusions</b>: Based on the evaluated parameters, the MPXV-2-Beta designed in this study exhibited significant potential as an effective candidate against hMPXV. This study establishes a foundation for developing an efficient vaccine against hMPXV, requiring further experimental and clinical validation to confirm computational findings.