严重急性呼吸综合征冠状病毒2（Severe Acute Respiratory Syndrome Coronavirus 2，SARS-CoV-2）感染引发的新型冠状病毒肺炎（Coronavirus Disease 2019，COVID-19）在全球范围内迅速蔓延，对人类健康和全球经济造成了重大威胁。随着疫苗的大规模接种以及病毒变异株的不断出现，人群免疫背景逐渐复杂化。然而，不同免疫背景人群的适应性免疫反应特征尚未完全阐明，这对疫苗的优化和免疫策略的制定提出了关键挑战。因此，综合考虑抗原暴露方式（如原型株/Omicron变异株感染和疫苗接种）与临床症状等因素，本研究旨在评估不同免疫背景人群中抗体动态变化及其对不同变异株的交叉反应性。此外，在Omicron变异株流行背景下，本研究将进一步探讨SARS-CoV-2原型株感染和（或）疫苗接种所形成的免疫印记（Immune Imprinting）对Omicron变异株感染后免疫反应的影响。为未来新发突发传染病的精准防控及疫苗的优化设计提供科学依据。

本研究基于新冠肺炎康复者随访队列，纳入了2020年1月至5月武汉市金银潭医院出院的第一波疫情期间的SARS-CoV-2感染者，并分别于2020年12月至2021年1月、2021年11月至2022年1月以及2023年2月至3月进行了三次连续随访。同时，纳入第一波疫情期间未感染SARS-CoV-2的武汉市社区人群作为对照。在一年随访时，所有研究对象均未接种新冠病毒疫苗，亦无SARS-CoV-2原型株或变异株的再感染；至两年随访时，约82.2%的新冠肺炎康复者已接种疫苗，但仍未发现SARS-CoV-2原型株或变异株的再感染；至三年随访时，约85.3%的康复者已接种疫苗，然而，随着疫情防控措施的调整，高达91.6%的康复者发生了Omicron变异株的再感染。

通过二元Logistic回归模型分析新冠肺炎康复者有症状再感染的危险因素。结果显示，与无基础疾病的康复者相比，合并基础疾病的康复者在一年随访时发生Omicron变异株有症状再感染的风险增加0.93倍（OR=1.93，95% CI: 1.22–3.07）；至两年随访时，风险增加1倍（OR=2.00，95% CI: 1.26–3.17）。与完全康复的个体相比，自然感染一年后仍存在长新冠症状的康复者发生Omicron变异株有症状再感染的风险增加1.49倍（OR=2.49，95% CI: 1.59–3.91）；自然感染两年后仍存在长新冠症状的康复者发生Omicron变异株有症状再感染的风险增加1.05倍（OR=2.05，95% CI: 1.34–3.13）。

采用间接酶联免疫吸附试验（Enzyme Linked Immunosorbent Assay，ELISA）、假病毒中和实验及荧光素酶报告基因系统等方法，分析不同免疫背景人群的抗体动态变化及交叉反应性。结果显示，在无抗原再暴露的情况下，SARS-CoV-2原型株自然感染后一年、两年和三年期间，血浆IgG、抗体依赖性细胞毒性作用（Antibody Dependent Cell-Mediated Cytotoxicity，ADCC）、抗体依赖性细胞吞噬作用（Antibody Dependent Cellular Phagocytosis，ADCP）及中和抗体水平随时间逐渐衰减（P<0.05）。随着SARS-CoV-2变异株的出现，针对Omicron变异株各亚系的血浆IgG、ADCC、ADCP及中和抗体反应的交叉保护作用显著减弱甚至消失（P<0.05）。至三年随访时，大多数康复者已接种疫苗并发生Omicron变异株再感染，机体产生了强烈的抗体反应（两年vs三年中和抗体滴度：原型株1:152.60 vs 1:495.80，P<0.001；BA.5变异株1:39.87 vs 1:170.40，P<0.001）。Omicron变异株再感染诱导的针对BA.5变异株的抗体反应低于针对原型株的抗体反应（BA.5变异株vs原型株中和抗体滴度：1:170.40 vs 1:495.80，P<0.001），但仍可产生较强的针对BA.5变异株的交叉保护作用；而对于抗原距离较远的JN.1变异株，其交叉中和保护反应基本消失（BA.5 vs JN.1中和抗体滴度：1:170.40 vs 1:37.21，P<0.001）。虽未能诱导出有效的针对JN.1变异株的中和抗体，但机体成功诱导了针对JN.1变异株的ADCC和ADCP效应。

进一步运用竞争ELISA和流式细胞术分析免疫印记的血清学特征及记忆B细胞亚型，探讨不同免疫背景人群中SARS-CoV-2原型株感染和（或）疫苗接种后形成的免疫印记对Omicron变异株感染后免疫反应的影响。结果显示，在Omicron BA.5变异株再感染后，不同免疫背景的研究对象中均未检测到仅针对BA.5变异株的特异性S-和RBD-IgG抗体。与此同时，机体保留了大量仅识别原型株的抗体，以及能够同时识别原型株和BA.5变异株的交叉反应性抗体。对于SARS-CoV-2原型株感染和（或）疫苗接种者，BA.5变异株再感染后检测到靶向原型株与BA.5变异株保守表位的交叉反应性记忆B细胞，而未检测到仅识别Omicron变异株的记忆B细胞；但对于仅接种疫苗者，在感染BA.5变异株后，机体则产生了特异性针对BA.5变异株的记忆B细胞。

综上，本研究系统揭示新冠肺炎康复者发生Omicron变异株有症状再感染的危险因素、不同免疫背景人群的抗体反应及Fc效应的动态变化及交叉反应特征、以及免疫印记对变异株再感染后的免疫反应的影响，为理解长期免疫保护机制提供了重要科学依据。研究明确了合并基础疾病者及存在长新冠症状的人群具有较高的再感染风险，为制定高风险人群的差异化防控策略提供了关键支持。研究进一步阐明了不同免疫背景人群中抗体反应和Fc效应的动态变化特征及针对Omicron不同亚系的交叉反应差异。值得注意的是，对于曾自然感染SARS-CoV-2原型株或接种疫苗的个体，在后续感染变异株后，其抗体反应和记忆B细胞主要针对原型株与变异株的保守表位。这一现象表明，由于免疫印记的存在，宿主倾向于优先激活能够快速且高效响应变异病毒的B细胞亚群，从而有效应对变异株的感染。上述研究为优化疫苗策略提供了依据，并强调了开发针对变异株的更新疫苗或广谱疫苗的重要性。本研究不仅为新冠肺炎疫情防控提供了重要参考，更为未来应对新发突发传染病的精准防控体系建设及疫苗优化设计提供了理论支持。

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has spread rapidly worldwide, posing significant threats to global public health and economic development. With the mass vaccination campaigns and continuous emergence of viral variants, the immunological profiles of populations have become increasingly complex. However, the characterization of adaptive immune responses in individuals with different immunological backgrounds remain incompletely understood, presenting critical challenges for vaccine optimization and immunization strategy development. By comprehensively evaluating factors including antigen exposure history (such as prototype strain/Omicron variant infection and vaccination) and clinical manifestations, this study aims to assess the dynamic changes in antibody responses across populations with varying immune backgrounds and their cross-reactivity against different viral variants. Furthermore, in the context of Omicron variant predominance, we will investigate how immune imprinting - established through prior SARS-CoV-2 prototype strain infection and/or vaccination - influences immune responses following Omicron variant infection. These findings will provide crucial scientific evidence to inform precision prevention strategies for future emerging infectious diseases and guide the design of optimized vaccination approaches.

This longitudinal study was conducted within a COVID-19 convalescent cohort comprising SARS-CoV-2 infected individuals discharged from Wuhan Jinyintan Hospital during the first wave of the pandemic (January-May 2020). Participants underwent three consecutive follow-up investigations: December 2020-January 2021 (1-year), November 2021-January 2022 (2-year), and February-March 2023 (3-year). A control group of Wuhan community residents without SARS-CoV-2 infection during the initial outbreak was simultaneously enrolled. At the 1-year follow-up, none of the study participants had received COVID-19 vaccination or experienced reinfection with either the SARS-CoV-2 prototype strain or its variants. By the 2-year follow-up, approximately 82.2% of the COVID-19 convalescents had been vaccinated against SARS-CoV-2, yet no cases of reinfection with the prototype strain or its variants were detected. At the 3-year follow-up, about 85.3% of the convalescents had received COVID-19 vaccination. However, following the comprehensive adjustment of epidemic prevention measures, a striking 91.6% of the convalescents experienced reinfection with Omicron subvariants.

We employed binary Logistic regression models to identify risk factors for symptomatic Omicron reinfection among COVID-19 convalescents. The analysis revealed that compared to recovered individuals without underlying conditions, those with comorbidities demonstrated a 0.93-fold (OR=1.93，95% CI: 1.22–3.07) increased risk of symptomatic Omicron reinfection at the 1-year follow-up, with this risk elevation reaching 1-fold (OR=2.00，95% CI: 1.26–3.17) by the 2-year follow-up. Notably, convalescents experiencing long COVID symptoms one year post-natural infection showed a 1.49-fold (OR=2.49，95% CI: 1.59–3.91) higher risk of symptomatic Omicron reinfection relative to fully recovered individuals, while those with persistent long COVID symptoms at two years post-infection maintained a 1.05-fold (OR=2.05，95% CI: 1.34–3.13) elevated reinfection risk.

We comprehensively analyzed antibody dynamics and cross-reactivity in individuals with varying immune backgrounds using indirect Enzyme Linked Immunosorbent Assay (ELISA), pseudovirus neutralization assays, and luciferase reporter gene systems. Longitudinal data demonstrated that without antigen re-exposure, plasma IgG levels, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and neutralizing antibody titers progressively declined (P<0.05) over one to three years following natural infection with the SARS-CoV-2 prototype strain. Notably, cross-protective antibody responses against Omicron subvariants were significantly diminished or lost (P<0.05). By the three-year follow-up, most convalescents had received vaccination and experienced Omicron reinfection, eliciting robust antibody responses (neutralizing antibody titers at two vs three years: prototype strain 1:152.60 vs 1:495.80, P<0.001; BA.5 variant 1:39.87 vs 1:170.40, P<0.001). While Omicron reinfection induced lower neutralizing responses against BA.5 variant compared to prototype strain (BA.5 vs prototype: 1:170.40 vs 1:495.80, P<0.001), substantial cross-protection against BA.5 variant was maintained. However, neutralization against the antigenically distant JN.1 variant was virtually abolished (BA.5 vs JN.1: 1:170.40 vs 1:37.21, P<0.001). Importantly, despite ineffective JN.1 variant neutralization, significant ADCC and ADCP responses against JN.1 variant were successfully induced, suggesting alternative protective mechanisms.

To further investigate immune imprinting characterization, we employed competitive ELISA and flow cytometry to analyze serological profiles and memory B cell subsets, examining how prior SARS-CoV-2 prototype strain infection and/or vaccination shaped immune responses following Omicron variant infection. Our findings revealed that following Omicron BA.5 variant reinfection, no BA.5-specific S-IgG or RBD-IgG antibodies were detected in any study participants across different immunological backgrounds. Meanwhile, the immune system retained substantial quantities of antibodies exclusively recognizing the prototype strain, along with cross-reactive antibodies capable of binding both the prototype strain and BA.5 variant. In individuals with prior SARS-CoV-2 prototype strain infection and/or vaccination, BA.5 variant reinfection elicited cross-reactive memory B cells targeting conserved epitopes shared between the prototype strain and BA.5 variant, but failed to induce BA.5-specific memory B cells. In contrast, vaccine-only recipients who experienced BA.5 infection developed memory B cells exclusively recognizing the BA.5 variant.

In summary, this study systematically elucidates the risk factors for symptomatic Omicron reinfection in COVID-19 convalescents, characterizes the dynamic changes of antibody responses and Fc-mediated effector functions across different immune backgrounds, and delineates how immune imprinting shapes post-reinfection immunity—providing crucial scientific insights into long-term immune protection mechanisms. Our findings identify individuals with comorbidities and persistent long COVID symptoms as high-risk populations for reinfection, offering critical evidence for developing targeted prevention strategies. The study further elucidates the dynamic characteristics of antibody responses and Fc effector functions in populations with different immunological backgrounds, as well as the differences in cross-reactivity against various Omicron sublineages. The study further elucidates the dynamic characteristics of antibody responses and Fc effector functions across diverse immune backgrounds, as well as their cross-reactive differences against various Omicron sublineages. Notably, in individuals with prior natural infection by the prototype SARS-CoV-2 strain or vaccination, subsequent variant infections elicited antibody responses and memory B cells predominantly targeting conserved epitopes shared between the prototype strain and variants. This phenomenon demonstrates that due to immune imprinting, the host preferentially activates pre-existing B cell subsets capable of rapid and efficient responses against viral variants, thereby effectively combating variant infections. These findings provide a scientific basis for optimizing vaccination strategies and underscore the critical need for developing updated variant-specific or broad-spectrum vaccines. The study not only offers valuable insights for COVID-19 pandemic control, but also provides theoretical support for establishing precision prevention systems and optimizing vaccine design against future emerging infectious diseases.