Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, MD, 21201, USA.
Department of Pathogen Biology, School of Basic Medical Sciences, China Medical University, Shenyang, 110122, China.
Microb Cell Fact. 2023 Sep 25;22(1):194. doi: 10.1186/s12934-023-02200-4.
Neutralizing antibody plays a key role in protecting hosts from invasive pathogens and their virulent components. Current high-throughput assays for antibody screening are based on binding activities. However, those antibodies with high affinity may not have neutralizing activities. Subsequent functionality assays are necessary to identify neutralizing antibodies from binders with high affinity to their target antigens, which is laborious and time-consuming. Therefore, a versatile platform that can rapidly identify antibodies with both high binding affinity and neutralizing activity is desired to curb future pandemics like COVID-19.
In this proof-of-concept study, we adapted Saccharomyces cerevisiae to either display human antibodies on the yeast surface or secrete soluble antibodies into the cultivation supernatant under a controllable 'switch' through different carbon source induced promoters. Initially, an engineered chimeric-bispecific Fab antibody, derived from humanized nanobodies against both Clostridioides difficile toxin A and B (TcdA and TcdB), was successfully expressed either on the yeast cell surface or in the culture medium with intact bioactivity, suggesting the applicability of our system in antibody display and secretion. Next, a combinatorial Fab library was constructed from B cells isolated from a convalescent patient with a high serological neutralizing titer against TcdB. Following three rounds of magnetic bead enrichment and one round of flow cytometry sorting, antibodies against TcdB were enriched efficiently. We then sorted out single binders with high binding affinity and induced them to express soluble antibodies in culture medium. The neutralizing activity of culture supernatant was analyzed using cell-based assay immediately. This way, we rapidly identified two unique neutralizers (out of seven binders) that can neutralize the cytotoxicity of TcdB.
The antibody screening platform described here simplifies the neutralizing antibody discovery procedure and will be an attractive alternative for screening functional antibodies against infectious diseases.
中和抗体在保护宿主免受入侵病原体及其毒力成分的侵害方面起着关键作用。目前用于抗体筛选的高通量检测方法基于结合活性。然而，那些具有高亲和力的抗体可能没有中和活性。需要进行后续的功能检测，才能从与目标抗原具有高亲和力的结合物中鉴定出中和抗体，这是一项繁琐且耗时的工作。因此，需要一种多功能平台，能够快速识别具有高结合亲和力和中和活性的抗体，以遏制像 COVID-19 这样的未来大流行。
在本概念验证研究中，我们通过不同碳源诱导的启动子，将酿酒酵母改造为在酵母表面展示人源抗体或分泌可溶性抗体到培养上清液中的两种形式。最初，通过人源化纳米抗体对艰难梭菌毒素 A 和 B（TcdA 和 TcdB）的亲和力，成功地在酵母细胞表面或培养上清液中表达了一种工程化的嵌合双特异性 Fab 抗体，且具有完整的生物活性，这表明我们的系统在抗体展示和分泌方面具有适用性。接下来，我们从一位具有高血清中和 TcdB 滴度的恢复期患者的 B 细胞中构建了一个组合 Fab 文库。经过三轮磁珠富集和一轮流式细胞术分选，有效地富集了针对 TcdB 的抗体。然后，我们从这些抗体中挑选出具有高结合亲和力的单克隆抗体，并诱导其在培养基中表达可溶性抗体。立即使用基于细胞的测定法分析培养上清液的中和活性。通过这种方式，我们快速鉴定出两种独特的中和剂（在七个结合物中），它们能够中和 TcdB 的细胞毒性。
这里描述的抗体筛选平台简化了中和抗体的发现过程，将成为筛选针对传染病的功能性抗体的一种有吸引力的替代方法。
