2019年底，新型冠状病毒（SARS-CoV-2）被发现后快速传播至世界各地，使全球公共卫生体系面临严峻考验。SARS-CoV-2所引起的疾病称为新型冠状病毒肺炎，随着疫情的持续，SARS-CoV-2突变体正在不断涌现。病毒刺突蛋白（Spike, S）不仅可以识别ACE2受体，介导病毒进入宿主细胞，也可诱导机体产生保护性免疫反应，更是单抗作用的主要靶标。因此，Spike蛋白上的位点突变对于病毒的感染性和抗原性具有巨大影响。此外，SARS-CoV-2的Spike蛋白质单体上存在22个潜在的N-糖基化位点，糖基化对Spike蛋白质折叠和形成三聚体至关重要，但这些糖基化位点的具体功能还不明确。上述这两个重要的科学问题，还未被详细研究。本课题对截至2020年5月6日，全球共享所有流感数据倡议中的SARS-CoV-2序列进行下载，过滤掉不完整、冗余和模糊的序列，进行下一步研究。

本课题构建基于水泡性口炎病毒（Vesicular stomatitis virus, VSV）载体的假病毒，对Spike蛋白上的突变位点的感染性和抗原性进行系统研究。共构建了106株突变体假病毒，包括51株RBD区位点突变、29株RBD区以外的重要位点突变以及26株糖基化相关位点突变。使用突变体假病毒感染293T-hACE2、Huh7、Vero、LLC-MK2等敏感细胞系，分析突变体的感染性变化。同时，使用突变体假病毒对13个单抗和10份新冠恢复期血清进行中和反应，进一步分析突变体的抗原性变化。

对51株RBD区位点突变的研究发现，RBD区的突变对单抗和恢复期血清的抗原性变化较大。其中A475V、F490L、V483A、L452R、Y508H、N439K、D614G+I472V和D614G+A435S这8个突变株明显降低了单抗的中和活性。截止到2021年5月16日，包含N439K、L452R、A475V等突变的序列均已超过100条，我们应该警惕这些突变可能具有潜在的对临床所用单抗的逃逸。此外，RBD区还存在多个突变株假病毒对单抗或恢复期的敏感性增强，这些位点包括N354D、F338L、V367F、Q409E、Q414E、I468F、I468T、Y508H和A522V等。推测这些位点突变使得Spike蛋白上的抗体识别表位得到更好的暴露，从而更容易被抗体结合。

对29株RBD区以外的重要位点突变的研究发现，D614G突变株及其联合突变株感染性增强10倍左右，这是本课题发现的唯一能够显著增强病毒感染性的位点。进一步分析，发现D614G突变株感染性增强的原因是由于病毒颗粒上Spike蛋白表达量增多，且S1蛋白在病毒表面更加稳定，不容易脱落。此外，A831V突变株假病毒对单抗B38和恢复期血清的敏感性降低。

对26株糖基化相关位点突变的研究发现，糖基化缺失大都降低了假病毒的感染性，其中N122Q、N343Q、N717Q、T719A、N801Q、N1074Q和N331Q+N343Q联合突变这7株突变体假病毒的感染性明显下降。N234Q糖基化缺失突变对157、247、B38、CB6、P2C-1F11、AB35、H014和H00S022这8株单抗的敏感性均出现了不同程度的降低，说明N234位的糖基化对于这8株单抗对抗原表位的识别至关重要。此外，N165Q糖基化缺失突变株对P2B-2F6单抗和部分恢复期血清更加敏感，N709Q糖基化缺失突变株对H014单抗和部分恢复期血清更加敏感。说明N165和N709位的糖基化遮蔽了Spike蛋白上的表位，糖基化缺失后可更充分的暴露抗体的识别表位。

随着时间的推移，预计SARS-CoV-2将在人群中继续进化。本课题提示科研工作者应密切关注病毒变异，尤其是影响病毒特性的突变。进一步指导未来疫苗开发、抗病毒药物和单抗的研究，最终达到更好的预防和治疗新型冠状病毒肺炎的目的。

At the end of 2019, SARS-CoV-2，which is causative agent of Coronavirus disease 2019, was discovered and quickly spread around the world, posing a severe threat to the global public health system. With the continuation of the COVID-19 epidemic, SARS-CoV-2 mutants are emerging. Spike protein can not only recognize ACE2 receptor and mediate virus entry into host cells, but also induce protective immune response, which is the main target of monoclonal antibody. Therefore, the site mutation of Spike protein has a great influence on infectivity and antigenicity of the virus. In addition, there are 22 potential N-glycosylation sites on Spike protein monomers. Glycosylation modification is very important for Spike protein folding and trimer formation, but the specific function of these glycosylation sites is not clear. These two important scientific issues have not been studied in detail. Therefore, this study downloads the SARS-CoV-2 sequences in Global Initiative of Sharing All Influenza Data as of May 6, 2020, filters out the incomplete, redundant, and fuzzy sequences, and then selects important sites for further research.

In this study, we systematically studied the infectivity and antigenicity of the mutation sites on Spike protein by constructing pseudoviruses based on VSV. A total of 106 strains of pseudoviruses with mutants were constructed, including 51 strains of RBD region mutations, 29 strains of important site mutations outside the RBD region and 26 strains of glycosylation-related site mutations. The mutant pseudovirus was used to infect sensitive cell lines such as 293T-hACE2, Huh7, Vero, and LLC-MK2, and the infectious changes of the mutants were analyzed. At the same time, 13 mAbs and 10 COVID-19 convalescent sera were tested by mutant pseudovirus, and the antigenicity of the mutant was further analyzed.

In the study of 51 strains of RBD site mutations, it was found that the RBD region mutations changed greatly on the antigenicity of monoclonal antibodies and convalescent sera. Among them, eight mutants, A475V, F490L, V483A, L452R, Y508H, N439K, D614G+I472V, and D614G+A435S, significantly decreased the neutralizing activity of monoclonal antibodies. As of March 4, 2021, there are more than 100 sequences containing N439K, L452R, A475V and other mutations. We should be vigilant that these mutations may have potential escape from clinical mAbs. In addition, there were several pseudoviruses in RBD region with increased sensitivity to monoclonal antibodies or convalescence, including N354D, F338L, V367F, Q409E, Q414E, I468F, I468T, Y508H and A522V. It is speculated that the mutations of these sites make the epitopes better exposed and thus more likely to be bound by antibodies.

Based on the study of the important site mutations outside the RBD region of 29 strains, it was found that the infectivity of the D614G mutant and its combined mutants increased about 10 times, which was the only site found to significantly enhance the infectivity of SARS-CoV-2. Further analysis showed that the increased infectivity of D614G mutant was due to the increased expression of Spike protein on the virus particles, and S1 protein was more stable on the virus surface and not easy to fall off. In addition, the sensitivity of A831V mutant pseudovirus to monoclonal antibody B38 and convalescent serum decreased.

Based on the study of 26 strains of glycosylation-related site mutations, it was found that most of the glycosylation deletions reduced the infectivity of pseudoviruses, and the infectivity of N122Q, N343Q, N717Q, T719A, N801Q, N1074Q and N331Q+N343Q mutants decreased significantly. The sensitivity of N234Q glycosylation deletion mutation to 157, 247, B38, CB6, P2C-1F11, AB35, H014, and H00S022 mAbs decreased in varying degrees, indicating that the glycosylation modification of N234 site is very important for the recognition of antigenic epitopes of these eight mAbs. In addition, the N165Q glycosylated deletion mutant was more sensitive to P2B-2F6 monoclonal antibody and partial convalescent serum, while N709Q glycosylation deleted mutant was more sensitive to H014 monoclonal antibody and partial convalescent sera. It is suggested that the glycosylation modification of N165 and N709 obscures the epitopes on Spike protein, and the loss of glycosylation modification can more fully expose the recognition epitopes of the antibody.

Over time, it is expected that the SARS-CoV-2 will continue to evolve in the population. This study suggests that researchers should pay close attention to virus variation, especially those with functional changes. This study will guide the future vaccine development, antiviral drugs, and monoclonal antibody research, and finally achieve a better prevention and treatment of COVID-19.