已知TcdA和TcdB是艰难梭菌的主要毒力属性。因此,中和TcdA和TcdB活性可以被认为是针对艰难梭菌感染(CDI)的有效治疗方法。在这项工作中,我们利用噬菌体展示技术选择单链可变片段(scFv)片段作为噬菌体表面展示的重组抗体,专门针对原生TcdA,或TcdB(nTcdA和nTcdB),及其重组C端组合重复寡肽(CROP)结构域(rTcdA和rTcdB)。经过三轮生物淘选,通过酶联免疫吸附测定(ELISA)对大量与TcdA或TcdB表现出高反应性的噬菌体克隆进行定量.此外,通过细胞活力和中和测定来表征所选择的scFvs。免疫标记的基因表达,IL-8和TNF-α,通过RT-qPCR在处理的Caco-2细胞中检查。还通过分子对接鉴定了中和scFv的表位。完全正确,通过ELISA鉴定了18种scFv抗体(TcdA为7种,TcdB为11种)。在选定的scFvs中,TcdA的两个克隆(rA-C2,A-C9)和TcdB的三个克隆(rB-B4,B-F5,B-F11)在Caco-2和Vero细胞中表现出最高的中和活性。此外,抗TcdA和抗TcdB抗体的混合物显着降低了Caco-2细胞中TNF-α和IL-8的mRNA表达。分子对接表明,scFv与毒素之间的相互作用主要限于TcdA或TcdB的CROP结构域。我们的结果共同为使用噬菌体展示开发针对艰难梭菌毒素的中和scFvs提供了更多见解。需要进一步的研究来仔细评估scFvs作为使用动物模型和临床试验的CDI替代疗法的潜力。重要性通过中和抗体靶向艰难梭菌的主要毒素是CDI的新治疗方法。这里,我们报道了一组新的抗-TcdA(rA-C2,A-C9)和抗-TcdB(rB-B4,B-F5和B-F11)重组抗体片段(scFvs),使用噬菌体展示技术从TomlinsonI和J文库中分离.这些scFv抗体能够中和它们各自的毒素,并且在不同的体外模型中显示出作为针对艰难梭菌的TcdA和TcdB的潜在治疗剂的前景。此外,硅分析显示,至少有两种中和机制,在这项工作中,可以提出对分离的scFvs的抑制毒素的细胞表面结合和抑制毒素内化。这些发现为特定scFvs在体外水平上对艰难梭菌毒素的适用性提供了更多见解。然而,需要进一步的研究来评估这些scFvs在临床上作为CDI治疗治疗剂的潜在应用.
TcdA and
TcdB are known as the major virulence attributes of Clostridioides difficile. Hence, neutralizing the TcdA and
TcdB activities can be considered as an efficient therapeutic approach against C. difficile infection (CDI). In this work, we utilized phage display technique to select single-chain fragment variable (scFv) fragments as recombinant antibodies displayed on the surface of phages, which specifically target native TcdA, or
TcdB (nTcdA and nTcdB), and their recombinant C-terminal combined repetitive oligopeptide (CROP) domains (rTcdA and rTcdB). After three rounds of biopanning, abundance of phage clones displaying high reactivity with TcdA or TcdB was quantified through enzyme-linked immunosorbent assay (ELISA). Furthermore, selected scFvs were characterized by cell viability and neutralization assays. The gene expression of immunological markers, IL-8 and TNF-α, was examined in treated Caco-2 cells by RT-qPCR. The epitopes of neutralizing scFvs were also identified by molecular docking. Totally, 18 scFv antibodies (seven for TcdA and 11 for
TcdB) were identified by ELISA. Among selected scFvs, two clones for TcdA (rA-C2, A-C9) and three clones for
TcdB (rB-B4, B-F5, B-F11) exhibited the highest neutralizing activity in Caco-2 and Vero cells. Moreover, the cocktail of anti-TcdA and anti-TcdB antibodies notably decreased the mRNA expression of TNF-α and IL-8 in Caco-2 cells. Molecular docking revealed that the interaction between scFv and toxin was mostly restricted to CROP domain of TcdA or TcdB. Our results collectively provided more insights for the development of neutralizing scFvs against C. difficile toxins using phage display. Further research is needed to meticulously evaluate the potential of scFvs as an alternative treatment for CDI using animal models and clinical trials.IMPORTANCETargeting the major toxins of Clostridioides difficile by neutralizing antibodies is a novel therapeutic approach for CDI. Here, we report a panel of new anti-TcdA (rA-C2, A-C9) and anti-TcdB (rB-B4, B-F5, and B-F11) recombinant antibody fragments (scFvs) isolated from Tomlinson I and J libraries using phage display technique. These scFv antibodies were capable of neutralizing their respective toxin and showed promise as potential therapeutics against TcdA and TcdB of C. difficile in different in vitro models. In addition, in silico analysis showed that at least two neutralization mechanisms, including inhibiting cell surface binding of toxins and inhibiting toxin internalization can be proposed for the isolated scFvs in this work. These findings provide more insights for the applicability of specific scFvs toward C. difficile toxins at in vitro level. However, further research is required to evaluate the potential application of these scFvs as therapeutic agents for CDI treatment in clinical setting.