Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A³³ to K⁶⁹) and the first coiled-coil domain (L¹²¹ to K¹⁵⁸). Here, the phage display was used to develop a variable heavy chain domain (VHH) that can block receptor binding sites of recombinant NadA (rec-NadA). A phage library displaying VHH was panned against synthetic peptides (NadA-gd^A33−K69 or NadA-cc^L121−K158), gene encoding VHH was amplified from bound phages and re-cloned in the expression vector, and the soluble VHHs containing disulfide bonds were overexpressed in the SHuffle E. coli. From the repertoire of 96 clones, two VHHs (VHH_F3–binding NadA-gd^A33−K69 and VHH_G9–binding NadA-cc^L121−K158) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHH_F3 and VHH_G9 significantly reduced the adhesion of NM on hBMECs in situ and hindered the traversal of NM across the in-vitro BBB model. The work presents a phage display pipeline with a single-round of panning to select receptor blocking VHHs. It also demonstrates the production of soluble and functional VHHs, which blocked the interaction between NadA and its receptor, decreased adhesion of NM on hBMECs, and reduced translocation of NM across BBB in-vitro. The selected NadA blocking VHHs could be promising molecules for therapeutic translation.

奈瑟氏菌 粘附素A（NadA），一种表面粘附素 脑膜炎奈瑟菌（NM）与多种细胞类型相互作用，包括人脑微血管内皮细胞（hBMEC），并在发病机理中发挥重要作用。NadA的受体结合口袋位于球形头部结构域（A ³³至K ⁶⁹）和第一个螺旋线圈结构域（L ¹²¹至K ¹⁵⁸）中。在这里，噬菌体展示被用于开发可变重链结构域（VHH），该结构域可以阻断重组NadA（rec-NadA）的受体结合位点。展示了VHH的噬菌体文库针对合成肽（NadA-gd ^A33-K69或NadA-cc ^L121-K158），从结合的噬菌体中扩增出编码VHH的基因，并将其重新克隆到表达载体中，并且在SHuffle中过表达了含有二硫键的可溶性VHH 大肠杆菌。从96个克隆的库中，最终选择了两个VHH（VHH _F3结合NadA-gd ^A33-K69和VHH _G9结合NadA-cc ^L121-K158），因为它们消除了rec-NadA与细胞受体之间的相互作用。用VHH _F3和VHH _G9预孵育NM可以显着降低NM对hBMEC的粘附原位 并阻碍了NM穿越 体外BBB模型。该工作提出了具有单轮淘选的噬菌体展示管线，以选择受体阻滞性VHH。它也证明了可溶性和功能性VHHs的产生，其阻断了NadA与其受体之间的相互作用，降低了NM在hBMECs上的粘附，并减少了NM在BBB中的转运。体外。所选的NadA阻断VHHs可能是有前途的分子用于治疗性翻译。