One Health Center of Excellence for Research and Training, School of Public Health, Sun Yat-Sen University, Guangzhou, People's Republic of China.
National Medical Products Administration Key Laboratory for Quality Monitoring and Evaluation of Vaccines and Biological Products, Guangzhou, People's Republic of China.
Int J Nanomedicine. 2023 Jan 19;18:353-367. doi: 10.2147/IJN.S387160. eCollection 2023.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants have risen to dominance, which contains far more mutations in the spike protein in comparison to previously reported variants, compromising the efficacy of most existing vaccines or therapeutic monoclonal antibodies. Nanobody screened from high-throughput naïve libraries is a potential candidate for developing preventive and therapeutic antibodies.
Four nanobodies specific to the SARS-CoV-2 wild-type receptor-binding domain (RBD) were screened from a naïve phage display library. Their affinity and neutralizing activity were evaluated by surface plasmon resonance assays, surrogate virus neutralization tests, and pseudovirus neutralization assays. Preliminary identification of the binding epitopes of nanobodies by peptide-based ELISA and competition assay. Then four multivalent nanobodies were engineered by attaching the monovalent nanobodies to an antibody-binding nanoplatform constructed based on the lumazine synthase protein cage nanoparticles isolated from the (AaLS). Finally, the differences in potency between the monovalent and multivalent nanobodies were compared using the same methods.
Three of the four specific nanobodies could maintain substantial inhibitory activity against the Omicron (B.1.1.529), of them, B-B2 had the best neutralizing activity against the Omicron (B.1.1.529) pseudovirus (IC = 1.658 μg/mL). The antiviral ability of multivalent nanobody LS-B-B2 was improved in the Omicron (B.1.1.529) pseudovirus assays (IC = 0.653 μg/mL). The results of peptide-based ELISA indicated that LS-B-B2 might react with the linear epitopes in the SARS-CoV-2 RBD conserved regions, which would clarify the mechanisms for the maintenance of potent neutralization of Omicron (B.1.1.529) preliminary.
Our study indicated that the AaLS could be used as an antibody-binding nanoplatform to present nanobodies on its surface and improve the potency of nanobodies. The multivalent nanobody LS-B-B2 may serve as a potential agent for the neutralization of SARS-CoV-2 variants.
严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）奥密克戎变体已占据主导地位，其 Spike 蛋白中的突变数量远远超过之前报道的变体，从而降低了大多数现有疫苗或治疗性单克隆抗体的功效。从高通量原始文库中筛选出的纳米抗体是开发预防和治疗性抗体的潜在候选物。
从原始噬菌体展示文库中筛选出 4 种针对 SARS-CoV-2 野生型受体结合域（RBD）的纳米抗体。通过表面等离子体共振分析、替代病毒中和试验和假病毒中和试验评估它们的亲和力和中和活性。通过肽基 ELISA 和竞争试验初步鉴定纳米抗体的结合表位。然后，通过将单价纳米抗体附着在基于从亮氨酸合酶蛋白笼纳米颗粒（AaLS）中分离出的抗体结合纳米平台上，构建了 4 种多价纳米抗体。最后，使用相同的方法比较单价和多价纳米抗体之间的效力差异。
这 4 种特异性纳米抗体中的 3 种仍能对奥密克戎（B.1.1.529）保持显著的抑制活性，其中 B-B2 对奥密克戎（B.1.1.529）假病毒的中和活性最强（IC=1.658μg/ml）。多价纳米抗体 LS-B-B2 在奥密克戎（B.1.1.529）假病毒试验中的抗病毒能力有所提高（IC=0.653μg/ml）。肽基 ELISA 的结果表明，LS-B-B2 可能与 SARS-CoV-2 RBD 保守区域的线性表位反应，这将初步阐明其对奥密克戎（B.1.1.529）保持强大中和能力的机制。
本研究表明，AaLS 可作为抗体结合纳米平台，将纳米抗体展示在其表面，并提高纳米抗体的效力。多价纳米抗体 LS-B-B2 可能成为中和 SARS-CoV-2 变体的潜在药物。
