<p>Conservation of CTL epitopes.</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.

伴随刺突蛋白突变的新型冠状病毒（SARS-CoV-2）变异株快速涌现，大幅削弱了现有疫苗的防护效力，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），特异性靶向Alpha、Beta、Gamma及Omicron变异株刺突蛋白的突变位点。研究团队从美国国家生物技术信息中心（NCBI）、全球共享所有流感数据倡议（GISAID）以及通用蛋白质知识库（UniProt）获取序列数据，进而预测了一系列表位，涵盖线性B细胞表位、细胞毒性T淋巴细胞（CTL）表位、辅助性T淋巴细胞（HTL）表位与γ干扰素（IFN-γ）诱导表位；所有入选表位均经过严格筛选，具备高抗原性、良好可溶性、非致敏性与低毒性。上述表位可实现广泛的全球人群覆盖率：主要组织相容性复合体I类（MHC I）覆盖76.83%，MHC II类覆盖87.43%，联合表位总覆盖率达93.8%。研究团队通过引入人β防御素3、PADRE及50S核糖体蛋白L7/L12三种佐剂优化疫苗构建体，并采用AAY、GPGPG、EAAAK与KK四种连接肽连接各功能元件，以提升结构稳定性与免疫激活效果。本研究通过GenSmart™平台完成密码子优化，并借助二硫键设计工具2（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（评估均方根偏差RMSD、均方根波动RMSF、回旋半径以及分子力学/泊松玻尔兹曼表面积MMPBSA）在内的多项计算分析，验证了疫苗构建体与人中和抗体的稳定结合相互作用。通过C-IMMSIM平台开展的免疫应答模拟进一步证实，两款构建体可触发强烈的体液免疫与细胞免疫应答。为推动实际应用，本研究同时完成了适配原核表达系统的密码子优化。本研究凸显了持续基因组监测在追踪SARS-CoV-2进化过程中的关键作用，可为下一代疫苗的研发提供重要参考。不过本研究仅局限于计算预测结果，仍需通过实验验证以确认其实际效力。