Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

Abstract

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-gdA33−K69 or NadA-ccL121−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 (VHHF3–binding NadA-gdA33−K69 and VHHG9–binding NadA-ccL121−K158) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHHF3 and VHHG9 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.

Engineering the Single Domain Antibodies Targeting Receptor Binding Motifs Within the Domain III of West Nile Virus Envelope Glycoprotein

“…In our previous study, receptor binding motifs on the DIII of WNV E glycoprotein interacting with human brain microvascular endothelial cells (hBMCEs) were identified as DIII-1 G299−K307 and DIII-2 V371−R388 (Mertinkova et al, 2020). In the present study, sdAbs recognizing DIII-1 G299−K307 and DIII-2 V371−R388 were developed using the M13KO7 pIII Hyperphage system as described by us recently (Kulkarni et al, 2020). The resulting sdAbs were evaluated for toxicity, hemocompatibility, and ability to hinder the interaction between DIII and endothelial cells and neutralize entry of WNV-like particles (WNV-VLP) in the cultured cells.…”

“…Immunization and blood collection were performed following the guidelines of the EU animal welfare legislation and the University's ethical committee. The llama was immunized with rDIII of WNV along with the other antigens (rDIII of tick-borne encephalitis virus and NadA protein of Neisseria meningitidis) as described in our recent study (Kulkarni et al, 2020). The strategy of the immunization of the llama with multiple antigens was adopted from the protocol described by Pardon et al (2014) with some modifications.…”

“…Steps involving the isolation of peripheral blood mononuclear cells (PBMCs) from the blood of the immunized llama, amplification of VHH (variable domain of the heavy chain only antibodies, gene fragment encompassing framework 1framework 4 of sdAbs), its cloning into phagemid pJB12, and subsequent transformation of E. coli XL-1 blue (New England Biolabs, Frankfurt, Germany) to generate the VHH-E. coli library were performed exactly as described in our earlier publication (Kulkarni et al, 2020). Thus, the details are described in Supplementary Method 1.…”

“…After three washings, streptavidin-HRP conjugate (1:30,000 in TBST-20; Sigma-Aldrich) was added, and the membranes were incubated for 1 h. Subsequently, five washes with TBST-20 (5 min each) and one with TBS (5 min) were performed before incubating the membranes with a SuperSignal West Dura chemiluminescent substrate, and the signal was recorded on a C-DiGit Blot Scanner (Odyssey CLx, Cambridge, United Kingdom). Simultaneously, sdAbs raised against N. meningitides [VHH F3 (Kulkarni et al, 2020): non-related sdAbs; negative control] and hyperimmune serum of horse surviving natural WNV infection (positive control) spotted on NC membranes, were included in the assay to undergo incubations with DIII-1 G299−K307 or DIII-2 V371−R388 , streptavidin-HRP conjugate, and chemiluminescent substrate, followed by signal detection.…”

Applications of nanobodies in brain diseases

“…In a recent study, Nbs VHHG9 and VHHF3 targeting Neisseria adhesin A (NadA), which can antagonize the interaction between recombinant NadA and cell receptors, were developed. The preincubation of Neisseria meningitidis with VHHF3 and VHHG9 could significantly reduce the adhesion of neisseria meningitidis to human microvascular endothelial cells in situ and prevent it from crossing a BBB model (human BMECs) in vitro , providing a new treatment idea ( 127 ). Rabies is also an infectious disease in which the CNS is mainly affected.…”