背景和目的

由新型冠状病毒（Severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）引起的新型冠状病毒性肺炎（Coronavirus disease 2019，COVID-19）自2019年底爆发以来，迅速在全世界传播。对于此类全球范围内流行的新发突发传染性疾病，有效的治疗和干预措施显得尤为重要。恢复期患者的血浆中含有适应性免疫反应产生的中和抗体和非中和抗体，能够通过与病毒表面抗原或者病毒受体抗原结合阻止病毒侵入细胞或者抗体依赖的细胞毒作用对被病毒感染的细胞进行吞噬和杀伤。在新发病原体疫情早期，由于缺乏特异性疫苗和药物，恢复期血浆是重要的具有潜在效果的防控手段，但恢复期血浆防控新发病原体的安全性和有效性一直存在争议。新冠恢复期血浆疗法尚未得到广泛认可，恢复期血浆的发生和作用机制、有效成分均未得到明确解释。本研究目的在于通过对SARS-CoV-2康复者、感染者和疫苗接种者群体体液免疫反应的相关分析，分别探究病毒感染对体液免疫反应水平的影响情况及影响因素、疫苗接种对体液免疫反应水平的影响效果，阐明恢复期血浆抗病毒能力的发生发展特征，明确恢复期血浆作用有效性的影响因素，并对恢复期血浆中的特异性抗体序列进行鉴定和筛选，以确定其有效成分，为恢复期血浆的采集和治疗应用提供依据，有助于应对未来的新发突发病原体。

方法

1. 分别收集成年新冠患者恢复健康后短期（1-2个月）和长期（1-2年）的血液样本，通过实验室检测得到新冠特异性血浆抗体、中和抗体以及记忆B细胞结果，与康复者基本信息进行相关性分析。

2. 收集成年新冠感染者血液样本，通过实验室检测得到新冠特异性血浆抗体、中和抗体以及记忆B细胞结果，与感染者基本信息进行相关性分析。

3.分别收集新冠康复者、未感染SARS-CoV-2的健康者在接种疫苗前后的血液样本，通过实验室检测得到其新冠特异性血浆抗体、中和抗体以及记忆B细胞结果，各自进行组间及组内比较。

4. 收集新冠康复者血液样本，通过实验室检测得到新冠特异性血浆抗体、中和抗体以及记忆B细胞结果，比较筛选出抗体水平较高的受试者进行B细胞单细胞测序后进行生信分析及抗体序列筛选。

结果

1.受试新冠康复者在SARS-COV-2感染3周后，N特异性IgM抗体水平继续下降，并在6周后达到较低水平，而S蛋白-受体结合域（S-receptor binding domain，S-RBD）特异性和N特异性IgG抗体在症状出现4周后均持续上升；受试新冠康复者在SARS-CoV-2感染后的体温和发热持续天数与S-RBD特异性IgG抗体水平相关，症状出现体温超过38.5 ℃或持续发热超过3天的受试者血浆中的S-RBD特异性IgG抗体水平较高；受试新冠康复者在康复1年后，血浆中的新冠特异性IgG抗体和 N IgG抗体的水平均与其是否接种疫苗强相关，接种疫苗的受试者抗体水平显著提高；受试新冠康复者在康复1年后，针对野生型（Wild type，WT）毒株的特异性中和抗体水平与年龄或者核酸阳性持续时间均呈负相关。

2.新冠轻型受试患者和重型/危重型受试患者在新冠特异性RBD（Omicron BA.4/5）IgG抗体水平上的差异有统计学意义；受试新冠患者的新冠特异性血浆抗体水平与年龄中等程度相关；COVID-19重型/危重型受试患者新冠特异性血浆抗体与其疫苗接种情况存在密切关联性。

3.SARS-CoV-2 Delta和Omicron突变毒株都会对由SARS-CoV-2 WT或基于WT设计的新冠疫苗引起的个体的中和作用存在免疫逃逸现象；接种新冠疫苗是有意义的，可以增强个体的体液免疫应答，提升对SARS-CoV-2的中和效用。

4. 对7例血浆抗体水平较高的受试者样本进行单细胞测序，拟定理想克隆型筛选标准，经筛选最后得到12个克隆型。

结论

新冠康复者在症状出现后4周血浆抗体处于高水平，症状出现体温超过38.5 ℃或持续发热超过3天的康复者可能是更好的恢复期血浆捐献者。在SARS-CoV-2感染1年后，没有疫苗接种和二次感染发生的情况下，康复者体内仍存在一定水平的IgG抗体。新冠特异性抗体水平还与年龄和疫苗接种情况相关，接受新冠疫苗接种的个体也能作为高效价恢复期血浆的供者。本研究的结论对于未来的新发突发病原体的恢复期血浆疗法有借鉴意义。

Background and objective

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly around the world since the outbreak began in late 2019. Effective treatment and intervention measures are particularly important for these emerging infectious diseases that are circulating globally. The plasma of convalescent patients contains neutralizing and non-neutralizing antibodies produced by the adaptive immune response, which can phagocytic and kill virus-infected cells by binding to the surface antigen of the virus or the receptor antigen of the virus, or by antibody-dependent cytotoxic action. Due to the lack of specific vaccines and drugs, convalescent plasma is an important and potentially effective therapies of prevention and control of emerging pathogens in the early stage of the epidemic. However, the safety and effectiveness of convalescent plasma for prevention and control of emerging pathogens has been controversial. Convalescent plasma therapy has not been widely recognized, and the occurrence, mechanism of action and active components of convalescent plasma have not been clearly explained. The purpose of this study was to explore the influence of virus infection on humoral immune response level and influencing factors, and the effect of vaccination on humoral immune response level respectively through the correlation analysis of SARS-CoV-2 recovered patients, infected patients and vaccinees. This study is helpful to improve the occurrence and development characteristics of the antiviral ability of convalescent plasma, discuss the factors affecting the effectiveness of convalescent plasma, and identify and screen the specific antibody sequences in convalescent plasma to determine its effective components. Moreover, it can provide a basis for the collection and therapeutic application of convalescent plasma, and help to cope with future outbreaks of pathogens.

Method

1. Short-term (1-2 months) and long-term (1-2 years) blood samples were collected from adult COVID-19 patients after they returned to health, and the results of novel coronavirus specific plasma antibodies, neutralizing antibodies and memory B cells were obtained through laboratory testing, and the correlation analysis was conducted with the basic information of the recovered patients.

2. Collect blood samples of adult SARS-CoV-2 infected persons, obtain the results of novel coronavirus specific plasma antibodies, neutralizing antibodies and memory B cells through laboratory testing, and conduct correlation analysis with the basic information of infected persons.

3. Blood samples of COVID-19 patients and healthy people not infected with SARS-CoV-2 before and after vaccination were collected, and the results of COVID-19 specific plasma antibodies, neutralizing antibodies and memory B cells were obtained through laboratory detection, and inter-group and intra-group comparisons were made respectively.

4. Blood samples of recovered COVID-19 patients were collected and the results of COVID-19 specific plasma antibodies, neutralizing antibodies and memory B cells were obtained through laboratory testing. The subjects with high antibody levels were compared and screened for B-cell single-cell sequencing, and then biosignal analysis and antibody sequence screening were performed.

Results

1. The level of N-specific IgM antibody continued to decrease 3 weeks after infection with SARS-COV-2 and reached a lower level 6 weeks later, while the S-RBD (Receptor-binding domain) specificity and N-specific IgG antibody continued to increase 4 weeks after symptom onset. The body temperature and duration of fever after SARS-CoV-2 infection of COVID-19 patients were correlated with the level of S-RBD-specific IgG antibody. The level of S-RBD-specific IgG antibody in plasma of patients with temperature exceeding 38.5 ℃ or continuous fever for more than 3 days was higher. One year after recovery, the levels of COVID-19 specific IgG antibodies and N IgG antibodies in plasma were strongly correlated with vaccination, and the antibody levels in vaccinated subjects were significantly increased. After 1 year of recovery, the level of specific neutralizing antibodies against Wild type (WT) strains was negatively correlated with age or duration of positive nucleic acid.

2. There were statistically significant differences in COVID-19 specific RBD (Omicron BA.4/5) IgG antibody levels between mild COVID-19 patients and severe/critical COVID-19 patients; The level of COVID-19 specific plasma antibodies was moderately correlated with age. There is a close correlation between COVID-19 specific plasma antibodies and their vaccination status in severe/critical COVID-19 patients.

3. Both SARS-CoV-2 Delta and Omicron mutant strains exhibit immune escape from individual neutralization induced by SARS-CoV-2 WT or WT-based COVID-19 vaccine; Vaccination against COVID-19 is meaningful because it can enhance the humoral immune response of individuals and improve the neutralization effect of SARS-CoV-2.

4. Single cell sequencing was performed on 7 samples of subjects with high plasma antibody levels, and the ideal clonotype screening criteria were drawn up. Finally, 12 clonotypes were obtained after screening.

Conclusions

Patients who have recovered from COVID-19 have high levels of plasma antibodies 4 weeks after the onset of symptoms, and those who have a temperature of more than 38.5 ℃ during symptoms or a fever that lasts for more than 3 days may be better convalescent plasma donors. After 1 year of SARS-CoV-2 infection, in the absence of vaccination and in the case of secondary infection, some level of IgG antibody is still present in the recovered patients. SARS-CoV-2-specific antibody levels are also correlated with age and vaccination status, and individuals who receive vaccination can also act as high-potency convalescent plasma donors. The conclusion of this study has reference significance for the convalescent plasma therapy of emergent pathogens in the future.