Isolation and characterization of phage display-derived scFv antibodies against human parechovirus 1 VP0 protein

Abstract

Human parechoviruses (PeVs) are common viruses that are associated with a variety of diseases from mild gastrointestinal and respiratory symptoms to severe central nervous system infections. Until now there has not been antibodies for visualizing parechovirus infection. We used E. coli recombinant PeV-A1-VP0 protein as a target in phage display single chain variable fragment (scFv) antibody library panning. Three rounds of panning allowed identification and isolation of several candidate scFv clones, which tested positive in enzyme-linked immunosorbent assay (ELISA) against VP0. Three scFv clones (scFv-55, -59 and -71) with different CDR-3 sequences were further purified and tested in ELISA, Western blot and immunofluorescence microscopy (IFA) against a set of PeV-A1 isolates and a few isolates representing PeV types 2-6. In IFA, all three scFv binders recognized twenty PeV-A1 isolates. ScFv-55 and -71 also recognized clinical representatives of PeV types 1-6 both in IFA and in capture ELISA, while scFv-59 only recognized PeV-A1, -A2 and -A6. PeV-A1-VP0 (Harris strain) sequence was used to generate a peptide library, which allowed identification of a putative unique conformational antibody epitope with fully conserved flanking regions and a more variable core VVTYDSKL, shared between the scFv antibodies. Sequencing of the VP0 region of virus samples and sequence comparisons against parechoviral sequences in GenBank revealed 107 PeV-A1, -A3, -A8, -A17, -A (untyped) sequences with this exact epitope core sequence, which was most dominant among PeV-A1 isolates. These data suggest the first-time isolation of broad range phage display antibodies against human parechoviruses that may be used in diagnostic antibody development.

Figure 1

Time-resolved fluoroimmunoassay of selected scFv clones. Based on CDR-H3 sequences, a total of 11 clones were selected for secondary screening against VP0-GST and GST in a TRF-assay using europium chelate. In the assay, His-tag purified scFv clones expressed as a fusion to bacterial alkaline phosphatase, His-tag and FLAG tag were bound to biotinylated VP0-GST and biotinylated GST on 96 well streptavidin microtiter plate. Bound scFv-AP was detected with polyclonal Eu-labelled anti-alkaline phosphatase antibody and time-resolved fluorescence from europium was measured with Victor multilabel counter. The Y-axis represents the europium counts per second. The X-axis represents the different clones screened based on different CDR-H3 sequence.

Figure 2

Determination of binding properties of scFv antibodies by sandwich ELISA, Western blot and IFA. (a) Sandwich ELISA assay of scFv antibodies was performed against PeV-A1, VP0, cell control lysate and GST. 96-well plate was coated with scFv-AP antibodies (500 ng/well) followed by addition of PeV-A1 virus, VP0 protein, control lysate and GST protein. Rabbit anti-PeV antiserum (pAb-PeV-A1) was used as detector antibody. HRP-conjugated anti-rabbit secondary antibody was added followed by the substrate. The reaction was stopped with H₂SO₄ and OD₄₅₀ was measured with Victor multilabel counter. (b) Detection of PeV-A1-VP0 by Western blot. M: marker; lane 1: PeV-A1 (Harris strain); lane 2: VP0-GST; lane 3: Cell lysate; lane 4: GST. The molecular weight of PeV-A1-VP0 protein is approximately 37 kDa and that of VP0-GST fusion protein about 64 kDa. (c) Detection of PeV-A1 with scFv and rabbit anti-PeV-A1. The HT-29 cells were infected with PeV-A1 (Harris strain), fixed, permeabilized and stained with scFv antibodies and polyclonal anti-PeV-A1 antiserum as a control. Virus is shown as green, and nuclei stained with DAPI in blue. Stained cells were visualized using EVOS FL Auto imaging microscope (10 × magnification).

Figure 3

Detection of PeV-A1-infected HT-29 cells with scFv-71. HT-29 cells were mock-inoculated or inoculated with 19 clinical PeV-A1 isolates followed by staining with scFv-71 antibody and rabbit anti-FLAG antibody followed by anti-rabbit Alexa Fluor 488 secondary antibody. Nuclei were stained with DAPI. Stained cells were visualized using EVOS FL Auto imaging microscope (10 × magnification).

Figure 4

Detection of PeV types 1 to 6 using scFv-71 in IFA. HT-29 cells were mock-infected or infected with PeV types 1 to 6 and stained with scFv-71 antibody followed by staining with rabbit anti-FLAG antibody and anti-rabbit Alexa Fluor 488 secondary antibody. Nuclei were stained with DAPI. Stained cells were visualized using EVOS FL Auto imaging microscope (10 × magnification). Virus strains used in the panel: PeV-A1 “Harris”, PeV-A2 “Williamson”, PeV-A3 “152037”, PeV-A4 “K2511776”, PeV-A5 “20552322” and PeV-A6 “89”.

Figure 5

Consensus sequence alignment and location of the epitope on a parechovirus capsid. (a) Top-down surface view of PeV-1 structure (PDB: 4z92) asymmetric unit consisting of VP0 (blue), VP1 (magenta), and VP3 (grey) proteins. The core of the putative epitope is colored in green, while the residues of the conserved flanking regions that exhibit surface exposure are colored in orange. Structurally the epitope is located near the junction of the three capsid proteins, which is also structurally close to the RGD motif in VP1, known to be important for integrin binding. The highly conserved flanking regions of the epitope are likely important in stabilizing the base structure of the epitope, which itself has a higher degree of flexibility. (b) Sequence alignment of the VP0 residue range obtained from Pepscan’s CLIPS analysis for PeV-1–6 with residues numbered based on the PeV-A1 (Harris) prototype sequence (acc. no. L02971). The area boxed in red represents residues with surface exposure. The flanking regions of the core, VVTYDSKL, are seen highly conserved across different PeV type, while the core alignment itself shows higher flexibility.