DataSheet_11_Revelation of Potent Epitopes Present in Unannotated ORF Antigens of SARS-CoV-2 for Epitope-Based Polyvalent Vaccine Design Using Immunoinformatics Approach.xlsx

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kills thousands of people worldwide every day, thus necessitating rapid development of countermeasures. Immunoinformatics analyses carried out here in search of immunodominant regions in recently identified SARS-CoV-2 unannotated open reading frames (uORFs) have identified eight linear B-cell, one conformational B-cell, 10 CD4+ T-cell, and 12 CD8+ T-cell promising epitopes. Among them, ORF9b B-cell and T-cell epitopes are the most promising followed by M.ext and ORF3c epitopes. ORF9b40-48 (CD8+ T-cell epitope) is found to be highly immunogenic and antigenic with the highest allele coverage. Furthermore, it has overlap with four potent CD4+ T-cell epitopes. Structure-based B-cell epitope prediction has identified ORF9b61-68 to be immunodominant, which partially overlaps with one of the linear B-cell epitopes (ORF9b65-69). ORF3c CD4+ T-cell epitopes (ORF3c2-16, ORF3c3-17, and ORF3c4-18) and linear B-cell epitope (ORF3c14-22) have also been identified as the candidate epitopes. Similarly, M.ext and 7a.iORF1 (overlap with M and ORF7a) proteins have promising immunogenic regions. By considering the level of antigen expression, four ORF9b and five M.ext epitopes are finally shortlisted as potent epitopes. Mutation analysis has further revealed that the shortlisted potent uORF epitopes are resistant to recurrent mutations. Additionally, four N-protein (expressed by canonical ORF) epitopes are found to be potent. Thus, SARS-CoV-2 uORF B-cell and T-cell epitopes identified here along with canonical ORF epitopes may aid in the design of a promising epitope-based polyvalent vaccine (when connected through appropriate linkers) against SARS-CoV-2. Such a vaccine can act as a bulwark against SARS-CoV-2, especially in the scenario of emergence of variants with recurring mutations in the spike protein.

严重急性呼吸综合征冠状病毒2型（Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）每日在全球造成数千人死亡，因此亟需快速开发防控手段。本研究通过免疫信息学（immunoinformatics）分析，在新近发现的SARS-CoV-2未注释开放阅读框（unannotated open reading frames, uORFs）中搜寻免疫优势区域（immunodominant regions），共鉴定出8个线性B细胞表位（linear B-cell epitope）、1个构象B细胞表位（conformational B-cell epitope）、10个CD4+T细胞表位（CD4+ T-cell epitope）以及12个CD8+T细胞表位（CD8+ T-cell epitope），均为具有开发潜力的候选表位。其中，ORF9b的B细胞与T细胞表位潜力最为突出，其次为M.ext及ORF3c表位。研究发现，ORF9b40-48（CD8+T细胞表位）具有极高的免疫原性与抗原性，且等位基因覆盖率（allele coverage）最高；此外，该表位与4个高效CD4+T细胞表位存在序列重叠。基于结构的B细胞表位预测（structure-based B-cell epitope prediction）显示，ORF9b61-68为免疫优势区域，其与1个线性B细胞表位（ORF9b65-69）存在部分重叠。ORF3c的CD4+T细胞表位（ORF3c2-16、ORF3c3-17及ORF3c4-18）与线性B细胞表位（ORF3c14-22）也被鉴定为候选表位。同理，M.ext及7a.iORF1（与M蛋白和ORF7a存在序列重叠）蛋白也携带具有开发潜力的免疫原区域。结合抗原表达水平，最终筛选出4个ORF9b表位与5个M.ext表位作为强效候选表位。突变分析进一步证实，上述筛选得到的uORF来源强效表位可抵御频发突变。此外，4个由经典开放阅读框（canonical ORF）编码的N蛋白（N-protein）表位也被证实具有高效性。综上，本研究鉴定的SARS-CoV-2 uORF来源的B细胞与T细胞表位，结合经典开放阅读框来源的表位，可为开发基于表位的多价疫苗（polyvalent vaccine，通过合适接头连接各表位）提供理论支撑。此类疫苗可作为抵御SARS-CoV-2的有效屏障，尤其适用于刺突蛋白（spike protein）频发突变的变异株出现的场景。