嵌合RBD庫：一種嶄新的疫苗策略用於製造廣效冠狀病毒疫苗

Abstract

新冠肺炎自2019年大爆發以來，病毒持續變異，儘管臨床上已有許多疫苗及藥物來預防及治療，其傳播力及免疫逃脫能力都持續增強，至今仍是全球健康體系的一大挑戰。胺基酸變異和重組造成的冠狀病毒多樣性降低了疫苗的預防效果，尤其是近來的新冠狀病毒重組變異體XBB。XBB病毒是BJ.1 and BM.1.1.1重組產生的，在刺突蛋白的受體結合結構域（RBD）中發生了重組。這種變異具有免疫逃脫及傳播性增加的特性。因此，我們需要開發新型疫苗能對各種新冠狀病毒變種和未來的Sarbecovirus具有廣泛的保護力。本研究想要透過從五種Sarbecovirus（SARS-CoV、 Delta、Omicron BA.2、穿山甲和蝙蝠冠狀病毒）中建立嵌合受體結合結構域（RBD）庫來開發對Sarbecovirus的廣效疫苗。利用PCR的片段擴增以及Golden Gate組裝，此文庫理論上有3125個獨特的嵌合RBD變異體。這個策略能反映自然界觀察到的冠狀病毒重組事件。到目前為止，已經鑑定出757種獨特的嵌合RBD，而它們均對與人類血管緊張素轉換酶2（hACE2）受體表現出不同的親和力。在建構文庫後，透過流式細胞儀的初步體外篩選，嚴格篩選出134個能對於hACE2具有很強親和力的嵌合RBD候選物，為“嵌合RBD疫苗”。這些嵌合RBD疫苗進一步在小鼠體內進行了免疫試驗。以87種嵌合RBD疫苗免疫的初步篩選結果表明， 26種疫苗對BA.4/5假病毒表現出交叉反應。尤其這些疫苗缺乏BA.4/5 RBD序列，MU4、XE3、WZ3和FC4仍然可以產生交叉反應高於Delta疫苗。特別是，與親代疫苗相比，XE3疫苗針對Omicron BA.4/5變異株引發了顯著更高的中和抗體水平。這結果表示XE3對多種Sarbecovirus變體具有潛在的廣效保護能力。總之，本研究為開發具有廣效保護能力的下一代新冠肺炎疫苗提供了策略。血清庫和疫苗庫篩選策略能幫忙加速未來疫苗開發工作。我們的策略強調了嵌合RBD疫苗在廣效保護以及增強對不斷發展的Sarbecovirus變異株的效率方面的潛力，有助於為未來的流行病做好準備。

The COVID-19 outbreak, caused by SARS-CoV-2, emerged in 2019 and rapidly escalated into a global pandemic. Despite the development of numerous clinical vaccines and therapeutic drugs, the persistent evolution of the virus continues to pose significant challenges. The virus's ability to mutate enhances its transmissibility and enables immune evasion, maintaining COVID-19 as a critical global health issue. The diversity of coronaviruses, driven by amino acid variations and recombination events, undermines the effectiveness of existing vaccines, especially the recent new recombinant variant XBB. XBB variant, a recombinant of two BA.2 lineages (BJ.1 and BM.1.1.1), has a recombination breakpoint in the spike protein's receptor-binding domain (RBD). This variant has acquired characteristics such as increased immune evasion and transmissibility, raising concerns about the adaptability of current vaccines. As a result, there is an urgent need for novel vaccines with broad protection against diverse SARS-CoV-2 variants and future Sarbecoviruses. This study aimed to develop a pan-Sarbecovirus vaccine strategy by creating a chimeric receptor binding domain (RBD) library from five Sarbecoviruses: SARS-CoV, Delta, Omicron BA.2, Pangolin coronavirus, and bat coronavirus. Using PCR-based fragment amplification and golden-gate assembly, the generated library theoretically consists of 3125 unique chimeric RBD variants, reflecting natural recombination events observed in coronavirus. So far, 757 unique chimeric RBDs have been identified, each demonstrating distinct binding affinities for the human angiotensin-converting enzyme 2 (hACE2) receptor. Following library construction, rigorous screening identified 134 chimeric RBD candidates with very strong hACE2 binding affinities through initial in vitro evaluations using flow cytometry. These selected candidates, termed "chimeric RBD vaccines," underwent in vivo immunization trials in mice. Preliminary results from immunization with 87 chimeric RBD vaccines indicated that 26 exhibited cross-neutralization capabilities against BA.4/5 pseudoviruses in collected mouse sera. Interestingly, vaccine candidates MU4, XE3, WZ3, and FC4 demonstrated higher pseudovirus inhibition percentages than the Delta vaccine, despite lacking BA.4/5 RBD sequences. In particular, the XE3 vaccine elicited significantly higher levels of neutralizing antibodies against the Omicron BA.4/5 sublineages when compared with parental vaccines, suggesting potential broad-spectrum protection against diverse Sarbecovirus variants. In conclusion, this study presents a strategy for developing next-generation COVID-19 vaccines with broad-spectrum protection capabilities. Serum bank establishment and versatile screening tools represent significant advancements that can expedite future vaccine development efforts. Our strategy highlights the potential of chimeric RBD vaccines for broad-spectrum protection and enhanced efficacy against emerging and evolving Sarbecovirus variants, contributing to preparedness for future pandemics.