Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been spreading globally since the end of 2019. During this period, the virus has undergone mutations that led to the emergence of multiple variants with high transmissibility or capability of immune evasion. To prevent the spread of the virus, large-scale vaccination campaigns were conducted in many countries. However, as SARS-CoV-2 continues to mutate, the prevalent COVID-19 vaccines, which were primarily designed using the SARS-CoV-2 prototype (PT) and effectively safeguarded against early SARS-CoV-2 variants, have displayed considerably diminished efficacy against subsequent Omicron sub-variants, especially against the more recent BF.7, BQ.1.1, CH.1.1, XBB, and XBB.1.5 that has higher capability immune escape. Therefore, there is an urgent need to develop a new generation of broad-spectrum vaccines that can generate high levels of neutralizing antibodies against multiple SARS-CoV-2 variants. Previously, our group developed the ZF2001 vaccine with a tandem repeat homodimer of the SARS-CoV-2 receptor-binding domain (RBD), which has been approved in 4 countries and vaccinated over 300 million doses worldwide. To tackle the Omicron variant, we also upgraded the RBD homodimer of ZF2001 vaccine to a Delta-BA.1 chimeric heterodimer and developed protein subunit and mRNA vaccine that both demonstrated potent broad-spectrum immunogenicity against pre-Omicron variants as well as some Omicron sub-variants such as BA.1 and BA.2. With further evolution of Omicron, the prevalent XBB derivatives have displayed severe immune escape to the BA.1 immunogen. Therefore, we aim to further broaden the immunogenicity of RBD dimer vaccines by upgrading the Delta-BA.1 RBD dimer immunogen using the RBDs of more recent Omicron sub-variants. In this study, we developed six novel RBD dimer mRNA vaccines, each incorporating an RBD dimer consisting of Delta RBD or the RBD from specific Omicron sub-variants such as BA.1, BA.2, or BA.5. By systematically evaluating the expression and immunogenicity of these RBD dimer mRNA vaccines, we validated their immunogenicity and potential as booster shots following two doses of inactivated vaccine, as these vaccines demonstrated potent humoral and cellular immunogenicity against a wide range of SARS-CoV-2 variants. Notably, the neutralization antibodies elicited by Delta-BA.2 (DO) and Delta-BA.5 (DO) vaccines showed complementary spectra of neutralization. In other words, DO and DO are more effective against SARS-CoV-2 variants that emerged before BA.2 and after BA.5, respectively. Thus, we further designed a cocktail vaccine by mixing the DO and DO vaccines, aiming to achieve a more broad-spectrum immunogenicity. Neutralization data showed that the cocktail vaccine successfully expanded the spectrum of immunogenicity compared with the DO or DO vaccine. Moreover, by administering a third booster dose, the DO and DO + DO cocktail vaccines effectively stimulated high levels of neutralizing antibodies targeting CH.1.1, XBB, and XBB.1.5. These findings contribute to our understanding of the spectrum neutralization across diverse variants, offering insights for the development of broad-spectrum COVID-19 vaccines.

严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)是导致新冠病 毒病(coronavirus disease 2019, COVID-19)的病原体. 2019年末至今, SARS-CoV-2在全球范围流行, 氨基酸突变累积 产生多种具有较高传播力或较强免疫逃逸能力的变异株. 目前广泛使用的COVID-19疫苗大多基于SARS-CoV-2原 型株(prototype, PT)进行免疫原设计. 这些疫苗针对早期变异株具有较好的保护效果, 然而面对后续出现的Omicron 系列变异株, 以原型株为免疫原的疫苗保护效力显著下降, 特别是在面对免疫逃逸极强的BF.7、BQ.1.1、CH.1.1、 XBB及XBB.1.5等变异株时, 难以激发较高水平特异性中和抗体. 因此, 亟须研发针对多种SARS-CoV-2变异株产生 高效中和抗体的新一代广谱疫苗. 本研究基于SARS-CoV-2受体结合域(receptor-binding domain, RBD)重复串联二 聚体的构型, 设计了6种包含Delta RBD以及BA.1、BA.2和BA.5等Omicron亚型RBD的串联二聚体mRNA疫苗, 并 对其表达情况及免疫原性进行了系统性评价. 同时, 还验证了在两针灭活疫苗基础上以mRNA疫苗作为加强针的 序贯免疫效果. 假病毒中和数据显示, Delta-BA.2(DO2)与Delta-BA.5(DO5)RBD二聚体疫苗表现出较优的免疫原性, 且两者诱导的中和抗体分别针对BA.2之前和BA.5之后出现的变异株有较好的中和活性, 表现出一定的互补趋势. 因此, 我们设计了DO2和DO5混合免疫的策略, 实现了对二者优势的互补, 进一步扩宽了疫苗中和抗体谱. 在三剂 免疫的结果中, DO5及DO2+DO5免疫可产生针对CH.1.1、XBB及XBB.1.5的高水平中和抗体. 这些结果有助于我们 理解不同分支变异株中和抗体谱的变化规律, 并为广谱COVID-19疫苗设计提供了参考.