2022年5月开始，猴痘引起了全球大流行，受到了广泛关注。本次猴痘疫情主要影响同时患有人类免疫缺陷病毒（HIV）的人群。在这一人群中，免疫防御机制的有效性显著降低，这可能阻碍他们有效对抗猴痘病毒的能力。被动抗体疗法是治疗猴痘的一种可行选择，但由于正痘病毒双层包膜蛋白的多样性和复杂的病毒入侵机制，高效的中和抗体研究受到了阻碍。本研究聚焦于中和抗体开发，通过跨学科技术手段突破传统抗体研发瓶颈，为新发突发传染病防治提供新型候选药物及方法论创新。

在本论文的第一部分，我们致力于开发靶向痘病毒的高效广谱中和抗体。利用6种高度保守且曾被报道为中和抗体靶点或疫苗成分的猴痘病毒膜蛋白，在高通量的全合成噬菌体展示文库中筛选出了纳米抗体M1R-01，靶向M1R蛋白，并系统性的鉴定了其体外和体内的抗病毒活性。它能够在体外有效中和痘苗及猴痘病毒。在VACV-WR感染的Balb/c小鼠模型中，鼻腔给药Tri-M1R-01或腹腔给药Fc-M1R-01可100%保护小鼠免于死亡，并减轻了疾病程度，降低了病毒载量。此外，我们与专业团队合作解析了纳米抗体Nb-M1R复合物的晶体结构，揭示了该抗体的新型特异性结合表位。

在此工作的指导下，我们将具有更强中和能力的鼠源抗体7D11进行人源化改造，获得抗体POX1.1，其与7D11具有相似的结合模式，改造改善了其与M1R的亲和能力。对于痘苗病毒及猴痘病毒的中和能力也提升了6-16倍。在VACV-WR感染的小鼠模型中，随着给药时间的提前，POX1.1显示出愈加强大的保护能力，提前腹腔给药可以在较低剂量下完全保护小鼠免于死亡。在MPXV感染的小鼠模型中，POX1.1也表现出了良好的抗病毒效果，显著的降低了病毒载量。这项工作为治疗和预防正痘病毒感染提供了两株有希望的候选药物。

在本论文的第二部分，我们聚焦于计算机辅助设计与合成抗体学在沙贝病毒广谱中和抗体研究中的作用。沙贝病毒属的SARS-CoV和SARS-CoV-2 分别在2003和2019年引起人类疾病疫情大流行，造成了致命威胁。本次SARS-CoV-2的不断变异也引起了广泛的关注和担忧，持续进化导致其能够有效地逃避中和抗体药物和康复血浆的攻击。开发具有广谱中和沙贝病毒活性的抗体有助于抵御目前的冠状病毒的进化突变以及未来可能出现的动物源性冠状病毒跨物种传播。于是本研究建立了两种发现广谱沙贝病毒中和抗体的方法。

首先，选择具有较为理想广谱中和活性的抗体S2H97作为模板，通过计算设计来进行优化。共设计了35株抗体，鉴定发现大多数抗体对多种变异株的中和活性提高了1.4-222倍不等。分子动力学模拟表明，设计抗体与RBD之间建立了新的分子间相互作用。在第二轮优化中，我们将设计的轻、重链重组，获得抗体 AI-1028，这株抗体的五个CDR区都被优化改造。且对测试的所有沙贝病毒株都显示出了最佳的中和活性。

除此之外，合成纳米抗体库也是快速开发抗体的宝贵资源。我们采用了不同种属RBD作为抗原经过多轮筛选后获得了两种新型纳米抗体，均对检测的多种沙贝病毒表现出了广谱中和活性。这些发现不仅提供了潜在的泛中和药物，在新型病毒变异株或新的人畜共患病毒出现时快速优化现有抗体开辟了一条新的途径，也为快速开发针对具有高度可变特性的新兴病原体的抗体疗法提供了指导。

综合来说，当猴痘或新冠等突发传染病爆发时，通过建立的计算机辅助设计或合成抗体学的方式，本研究鉴定出了针对病原体的广谱中和抗体，为疾病的抗体治疗提供候选药物，同时也为应对突发传染病提供了可快速响应的技术平台。

A new round outbreak of mpox occurred in May 2022, precipitating a marked increase in global cases. particularly affecting individuals co-infected with HIV. In this population, compromised immune defenses hinder effective antiviral responses, necessitating passive antibody therapy. Despite this, the investigations of effective antibody therapeutics have been hindered by the varied nature of orthopoxvirus envelope proteins and the intricate mechanisms underpinning viral invasion.

In the first part of this thesis, we are committed to developing highly efficient and broad-spectrum neutralizing antibodies targeting poxviruses. We used six highly conserved mpox virus membrane proteins that were previously reported as neutralizing antibody targets or vaccine components to screen nanobodies from a high-throughput fully synthetic phage display library. Notably, we identified a cross-reactive Nb, termed M1R-01, which targets the M1R protein and effectively neutralizes both Vaccinia virus (VACV strain MVA and WR) and MPXV. The trivalent M1R-01 completely neutralized the vaccinia virus in vitro. Animal experiments showed that intranasal administration of Tri-M1R-01 or intraperitoneal injection of Fc-M1R-01 can protect Balb/c mice from lethal VACV challenge, demonstrating the ability to effectively inhibit virus replication and dissemination. Additionally, we collaborated with professional teams to elucidate the crystal structure of the Nb-M1R nanobody complex, revealing a novel specific binding site on the antibody that is different from the binding site of the previously reported highly effective neutralizing antibody 7D11.

Guided by this work, we humanized the murine antibody 7D11 with enhanced neutralizing capacity, generating the antibody POX1.1. POX1.1 maintains a binding mode similar to 7D11 while improving its affinity for M1R. The neutralization potency against vaccinia virus and monkeypox virus was increased several-fold. In a murine model of VACV-WR infection, POX1.1 demonstrated progressively stronger protective efficacy with earlier administration timing, achieving complete protection against lethality at lower doses when delivered via intraperitoneal injection in advance. In a murine model of MPXV infection, POX1.1 also demonstrated robust antiviral efficacy, significantly reducing viral load. This study provides two promising candidate agents for the treatment and prophylaxis of orthopoxvirus infections.

In the second part of this thesis, we focus on the role of computer-aided design and synthetic nanobody libraries in the research of broad-spectrum neutralizing antibodies against Sarbecoviruses. The subgenus Sarbecovirus includes human SARS-CoV, SARS-CoV-2, and hundreds of genetically related bat viruses. SARS-CoV and SARS-CoV-2 have twice caused deadly threats to humans in 2003 and 2019. There is increasing concern about the rapid mutation of SARS-CoV-2, the continuous evolution has led to the striking evasion of neutralizing antibody drugs and convalescent plasma. Antibodies with broad activity across sarbecoviruses would be helpful to combat current SARS-CoV-2 mutations and longer-term animal virus spillovers. For this purpose, we explored and established two methods for discovering pan-Sarbecorvirus neutralizing antibodies.

Firstly, we chosed S2H97, a previously reported RBD antibody with ideal breadth and resistance to escape, as a template for computational design to enhance the neutralization activity and spectrum. A total of 35 designs were purified for evaluation. The neutralizing activity of a large proportion of these designs against multiple variants was increased from several to hundreds of times. Molecular dynamics simulation suggested that extra interface contacts and enhanced intermolecular interactions between the RBD and the designed antibodies are established. After light and heavy chain reconstitution, AI-1028, with five complementarity determining regions optimized, showed the best neutralizing activity across all tested sarbecoviruses.

In addition to computational design, chemically synthesized nanobody libraries are also a precious resource for rapid antibody development.  By applying distinct RBDs as antigens for reciprocal screening, we identified two novel nanobodies with broad activities. These findings provide potential pan-sarbecovirus neutralizing drugs and highlight new pathways to rapidly optimize therapeutic candidates when novel SARS-CoV-2 escape variants or new zoonotic coronaviruses emerge. And provide guidance for the rapid development of antibody therapeutics against emerging pathogens with highly variable characteristics.

In summary, when outbreaks of infectious diseases such as monkeypox or COVID-19 occur, we identify broadly neutralizing antibodies against the pathogens through established computer-aided design or synthetic antibody approaches, providing candidate drugs for antibody therapy of the diseases and also offering a scalable technical framework for rapid response to future pandemics.