Vashisht Kapil, Goyal Bharti, Pasupureddy Rahul, Na Byoung-Kuk, Shin Ho-Joon, Sahu Dibakar, De Sajal, Chakraborti Soumyananda, Pandey Kailash C
Parasite-Host Biology, Indian Council of Medical Research-National Institute of Malaria Research (ICMR-NIMR), Delhi, IND.
Biological Sciences, Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, IND.
Cureus. 2023 Feb 10;15(2):e34827. doi: 10.7759/cureus.34827. eCollection 2023 Feb.
Background The nucleocapsid protein (N protein) of SARS-CoV-2 is undeniably a potent target for the development of diagnostic tools due to its abundant expression and lower immune evasion pressure compared to spike (S) protein. Methods Blood samples of active COVID-19 infections (n=71) and post-COVID-19 (n=11) were collected from a tertiary care hospital in India; pre-COVID-19 (n=12) sera samples served as controls. Real-time reverse transcriptase-PCR (rRT-PCR) confirmed pooled sera samples (n=5) were used with PEPperCHIP® SARS-CoV-2 Proteome Microarray (PEPperPRINT GmbH, Germany) to screen immunodominant epitopes of SARS-CoV-2. Highly immunodominant epitopes were then commercially synthesized and further validated for their immunoreactivity by dot-blot and ELISA. Results The lowest detectable concentration (LDC) of the N1 peptide in the dot-blot assay was 12.5 µg demonstrating it to be fairly immunoreactive compared to control sera. IgG titers against the contiguous peptide (N2: 156AIVLQLPQGTTLPKGFYAEGS176) was found to be significantly higher (p=0.018) in post-COVID-19 compared to pre-COVID-19 control sera. These results suggested that N2-specific IgG titers buildup over time as expected in post-COVID-19 sera samples, while a non-significant immunoreactivity of the N2 peptide was also observed in active-COVID-19 sera samples. However, there were no significant differences in the total IgG titers between active COVID-19 infections, post-COVID-19 and pre-COVID-19 controls. Conclusion The N2-specific IgG titers in post-COVID-19 samples demonstrated the potential of N protein as an exposure biomarker, particularly in sero-surveillance studies.
背景 严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的核衣壳蛋白（N蛋白）由于其表达丰富且与刺突（S）蛋白相比免疫逃逸压力较低，无疑是开发诊断工具的有力靶点。方法 从印度一家三级护理医院收集了活动性冠状病毒病2019（COVID-19）感染患者（n = 71）和COVID-19康复后患者（n = 11）的血样；COVID-19之前（n = 12）的血清样本用作对照。实时逆转录聚合酶链反应（rRT-PCR）确认的混合血清样本（n = 5）与PEPperCHIP® SARS-CoV-2蛋白质组微阵列（德国PEPperPRINT GmbH公司）一起用于筛选SARS-CoV-2的免疫显性表位。然后对高度免疫显性表位进行商业合成，并通过斑点印迹和酶联免疫吸附测定（ELISA）进一步验证其免疫反应性。结果 斑点印迹试验中N1肽的最低可检测浓度（LDC）为12.5μg，表明与对照血清相比，其具有相当强的免疫反应性。与COVID-19之前的对照血清相比，发现COVID-19康复后患者针对连续肽段（N2：156AIVLQLPQGTTLPKGFYAEGS176）的IgG滴度显著更高（p = 0.018）。这些结果表明，如预期的那样，COVID-19康复后血清样本中N2特异性IgG滴度随时间升高，而在活动性COVID-19血清样本中也观察到N2肽的免疫反应性不显著。然而，活动性COVID-19感染患者、COVID-19康复后患者和COVID-19之前对照患者之间的总IgG滴度没有显著差异。结论 COVID-19康复后样本中的N2特异性IgG滴度证明了N蛋白作为暴露生物标志物的潜力，特别是在血清学监测研究中。
