<p>Mutations and frequency related to them.</p>

The rapid emergence of SARS-CoV-2 variants with spike protein mutations undermines the effectiveness of current vaccines, necessitating innovative strategies to ensure broad and lasting immunity. This study leverages an immunoinformatics approach to design two multi-epitope vaccine constructs Cov19-B (649 amino acids, 74 kDa) and Cov19-T (465 amino acids, 48 kDa) specifically targeting mutations in the spike protein observed in the Alpha, Beta, Gamma, and Omicron variants. Using sequence data retrieved from NCBI, GISAID, and UniProt, we predicted a range of epitopes, including linear B-cell, cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and IFN-gamma-inducing epitopes, selected for their high antigenicity, solubility, non-allergenicity, and non-toxicity. These epitopes provide extensive global population coverage: 76.83% for MHC I, 87.43% for MHC II, and 93.8% for combined epitopes. The constructs were enhanced with adjuvants—Human Beta-defensin 3, PADRE, and 50S ribosomal protein L7/L12—and connected with AAY, GPGPG, EAAAK, and KK linkers to optimize structural stability and immune activation. Codon-optimized has done using GenSmart™, and structurally stabilized via disulfide engineering (Disulfide by Design 2). Computational analyses, including molecular docking and dynamics simulations (assessing RMSD, RMSF, gyration, and MMPBSA), validated stable binding interactions with human neutralizing antibodies. Immune response simulations conducted via C-IMMSIM further confirmed the constructs’ capacity to trigger robust humoral and cellular immunity. To enable practical application, codon optimization was performed for efficient expression in prokaryotic systems. This study highlights the vital role of continuous genomic surveillance in tracking SARS-CoV-2 evolution and informs the development of next-generation vaccines. However, the study is limited to computational predictions, requiring experimental validation to confirm efficacy.

随着携带刺突蛋白突变的SARS-CoV-2变异株快速出现，现有疫苗的保护效力不断受到削弱，亟需创新策略以实现广谱且持久的免疫保护。本研究采用免疫信息学（immunoinformatics）方法，设计了两款多表位疫苗候选构建体Cov19-B（含649个氨基酸，分子量74 kDa）与Cov19-T（含465个氨基酸，分子量48 kDa），其靶向Alpha、Beta、Gamma及Omicron变异株的刺突蛋白突变位点。研究团队从NCBI、GISAID及UniProt数据库获取序列数据，预测了一系列表位，包括线性B细胞表位、细胞毒性T淋巴细胞（cytotoxic T lymphocyte，CTL）、辅助性T淋巴细胞（helper T lymphocyte，HTL）以及干扰素-γ诱导表位，并筛选出抗原性高、溶解性好、无致敏性且无毒的表位。这些表位具备广泛的全球人群覆盖范围：MHC I类分子覆盖度达76.83%，MHC II类分子覆盖度达87.43%，联合表位覆盖度则为93.8%。本研究为该构建体添加了人β防御素3（Human Beta-defensin 3）、PADRE以及50S核糖体蛋白L7/L12作为佐剂，并通过AAY、GPGPG、EAAAK及KK连接肽进行连接，以优化其结构稳定性与免疫激活能力。本研究通过GenSmart™平台完成密码子优化，并借助二硫键工程技术（Disulfide by Design 2）实现结构稳定化。包括分子对接与分子动力学模拟（评估指标涵盖RMSD、RMSF、回旋半径及MMPBSA）在内的计算分析结果证实，该构建体可与人中和抗体形成稳定的结合相互作用。通过C-IMMSIM工具开展的免疫响应模拟实验进一步证实，两款构建体均可触发强烈的体液免疫与细胞免疫应答。为推动实际应用，研究团队针对原核表达系统完成了密码子优化以实现高效表达。本研究凸显了持续基因组监测在追踪SARS-CoV-2演化过程中的关键作用，并为下一代疫苗的研发提供了参考依据。不过，本研究仅局限于计算预测层面，仍需通过实验验证以确认其实际效力。