Tan Selene Si Ern, Tam Ee Hong, Lai Kah Man, Wu Yanjun, Xiao Tianshu, Tan Yee-Joo
Infectious Diseases Translational Research Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore.
School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
Vaccines (Basel). 2025 Jun 26;13(7):688. doi: 10.3390/vaccines13070688.
BACKGROUND/OBJECTIVES: For viral entry into host cells, the spike (S) protein of coronavirus (CoV) uses its S1 domain to bind to the host receptor and S2 domain to mediate the fusion between virion and cellular membranes. The S1 domain acquired multiple mutations as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved to give rise to Variant of Concerns (VOCs) but the S2 domain has limited changes. In particular, the stem helix in S2 did not change significantly and it is fairly well-conserved across multiple beta-CoVs. In this study, we generated a murine mAb 7B2 binding to the stem helix of SARS-CoV-2.
MAb 7B2 was isolated from immunized mouse and its neutralization activity was evaluated using microneutralization, plaque reduction and cell-cell fusion assays. Bio-layer interferometry was used to measure binding affinity and AlphaFold3 was used to model the antibody-antigen interface.
MAb 7B2 has lower virus neutralizing and membrane block activities when compared to a previously reported stem helix-binding human mAb S2P6. Alanine scanning and AlphaFold3 modeling reveals that residues K1149 and D1153 in S form a network of polar interactions with the heavy chain of 7B2. Conversely, S2P6 binding to S is not affected by alanine substitution at K1149 and D1153 as indicated by the high ipTM scores in the predicted S2P6-stem helix structure.
Our detailed characterization of the mechanism of inhibition of 7B2 reveals its distinctive binding model from S2P6 and yields insights on multiple neutralizing and highly conserved epitopes in the S2 domain which could be key components for pan-CoV vaccine development.
背景/目的：冠状病毒（CoV）的刺突（S）蛋白利用其S1结构域与宿主受体结合，并利用S2结构域介导病毒粒子与细胞膜之间的融合，从而进入宿主细胞。随着严重急性呼吸综合征冠状病毒2（SARS-CoV-2）进化产生关注变异株（VOCs），S1结构域出现了多个突变，但S2结构域变化有限。特别是，S2中的茎螺旋没有显著变化，并且在多种β冠状病毒中相当保守。在本研究中，我们制备了一种与SARS-CoV-2茎螺旋结合的鼠单克隆抗体7B2。
从免疫小鼠中分离出单克隆抗体7B2，并使用微量中和、蚀斑减少和细胞-细胞融合试验评估其中和活性。采用生物层干涉术测量结合亲和力，并使用AlphaFold3对抗体-抗原界面进行建模。
与先前报道的与茎螺旋结合的人单克隆抗体S2P6相比，单克隆抗体7B2具有较低的病毒中和活性和膜阻断活性。丙氨酸扫描和AlphaFold3建模显示，S中的K1149和D1153残基与7B2重链形成极性相互作用网络。相反，如预测的S2P6-茎螺旋结构中的高ipTM分数所示，S2P6与S的结合不受K1149和D1153处丙氨酸取代的影响。
我们对7B2抑制机制的详细表征揭示了其与S2P6不同的结合模式，并对S2结构域中的多个中和且高度保守的表位提供了见解，这些表位可能是泛冠状病毒疫苗开发的关键组成部分。
