Selection and identification of single-domain antibody against Peste des Petits Ruminants virus

Abstract

Background: Peste des petits ruminants (PPR) is an infectious disease caused by the peste des petits ruminants virus (PPRV) that mainly produces respiratory symptoms in affected animals, resulting in great losses in the world's agriculture industry every year. Single-domain variable heavy chain (VHH) antibody fragments, also referred to as nanobodies, have high expression yields and other advantages including ease of purification and high solubility.

Objectives: The purpose of this study is to obtain a single-domain antibody with good reactivity and high specificity against PPRV.

Methods: A VHH cDNA library was established by immunizing camels with PPRV vaccine, and the capacity and diversity of the library were examined. Four PPRV VHHs were selected, and the biological activity and antigen-binding capacity of the four VHHs were identified by western blot, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) analyses. ELISA was used to identify whether the four VHHs were specific for PPRV, and VHH neutralization tests were carried out. ELISA and western blot analyses were used to identify which PPRV protein was targeted by VHH2.

Results: The PPRV cDNA library was constructed successfully. The library capacity was greater than 2.0 × 10⁶ cfu/mL, and the inserted fragment size was approximately 400 bp to 2000 bp. The average length of the cDNA library fragment was about 1000 bp, and the recombination rate was approximately 100%. Four single-domain antibody sequences were selected, and proteins expressed in the supernatant were obtained. The four VHHs were shown to have biological activity, close affinity to PPRV, and no cross-reaction with common sheep diseases. All four VHHs had neutralization activity, and VHH2 was specific to the PPRV M protein.

Conclusions: The results of this preliminary research of PPRV VHHs showed that four screened VHH antibodies could be useful in future applications. This study provided new materials for inclusion in PPRV research.

Keywords: Peste des petits ruminants virus; cDNA library; single-domain antibody.

Fig. 1. Construction of cDNA library. (A) White light diagram of normal Vero cells and lesions after immunization with attenuated PPRV vaccine. (B) Enzyme-linked immunosorbent assay was used to detect the reaction of polyclonal serum to PPRV. At 21 days after the third immunization, samples were collected to determine the antibody titer. The x-axis shows dilutions from 1:500 to 1:16,000. PPRV was used as the coating antigen. Serum obtained before immunization was used as the negative control. (C) The total RNA extracted from peripheral blood lymphocytes was purified to obtain an electrophoretic map of the mRNA. Lane 1: mRNA, the three bands are 28s, 18S, and 5S from top to bottom. (D) Electrophoresis of double-stranded cDNA. Lane 1: double-stranded cDNA. (E) Electrophoresis of purified double-stranded cDNA. Lane 1: purified double-stranded cDNA. Lane M: 250 bp DNA Ladder.

PPRV, peste des petits ruminants virus.

Fig. 2. Identification of cDNA library. (A) Library diversity detection. Lanes 1–16: 16 of the monoclonal electrophoresis results were randomly selected after the library's construction. Lane M: 250 bp DNA Ladder. (B) Lane 1: plasmid electrophoresis results extracted from the library. Lane M: Lambda EcoT14 I digest.

Fig. 3. Alignments of the amino acid sequences of VHHs. Thick-lined boxes indicate the specific VHH amino acids, the CDRs are shown in thin-lined boxes.

FR, framework region; CDR, complementarity determining region; VHH, variable domain of heavy chain.

Fig. 4. Soluble analysis and expression of VHH proteins. (A) IPTG induces the expression of four proteins in E. coli. Lanes 1, 3, 5, 7: bacterial lysates of uninduced VHH1, VHH2, VHH3, and VHH4. Lanes 2, 4, 6, 8: bacterial lysates of VHH1, VHH2, VHH3, and VHH4 induced with IPTG. (B) Soluble expression analysis of VHH1 and VHH2. Lanes 1 and 5: bacterial lysates from groups VHH1 and VHH2 were not induced with IPTG. Lanes 2 and 5, expression of VHH1 and VHH2 in E. coli induced by IPTG. Lane 3, 4, 7, and 8, VHH1 and VHH2 supernatant and precipitated expression products after IPTG induction. (C) Soluble expression analysis of VHH3 and VHH4. Lanes 1 and 5: bacterial lysates from groups VHH3 and VHH4 were not induced with IPTG. Lanes 2 and 5, expression of VHH3 and VHH4 in E. coli induced by IPTG. Lane 3, 4, 7, and 8, VHH3 and VHH4 supernatant and precipitated expression products after IPTG induction. (D) Purification of VHH1 and VHH2 antibodies. Lanes 3 and 8, the supernatant of bacterial lysates of VHH1 and VHH2. Lanes 1 and 6, the supernatant of bacterial lysates combined with the purification column results of VHH1 and VHH2. (E) Lanes 2 and 8, the supernatant of bacterial lysates of VHH3 and VHH4. Lanes 1 and 6, the supernatant of bacterial lysates combined with the purification column results of VHH3 and VHH4. Lane M: Protein marker.

VHH, variable domain of heavy chain.

Fig. 5. Characterization and application of purified VHHs. (A) Western blot analysis was performed with HRP conjugate anti-His mouse monoclonal antibody. (B) The affinity of VHH1, VHH2, VHH3, and VHH4 with PPRV was determined by indirect ELISA. PPRV was used as an enzyme plate-coated antigen and was diluted at the ratio of 1: 80. Four single-domain antibodies were added to the plate as the primary antibody, and anti-His mouse monoclonal antibody was added as the secondary antibody. (C) Cross-reactions with other viral antigens. ELISA detected the specificity of the VHH antibodies. The antigens of sheep and GPV, ORFV, FMDV, PEDV, and PPRV were used to evaluate PPRV specificity. The OD₄₅₀ value was determined as the average of three wells. Error bars represent mean ± SD values.

VHH, variable domain of heavy chain; PPRV, peste des petits ruminants virus; ELISA, enzyme-linked immunosorbent assay; GPV, goat pox virus; ORFV, sheep mouth sore virus; FMDV, foot and mouth disease virus; PEDV, porcine epizootic diarrhea virus.

Fig. 6. The reaction of VHH antibodies with PPRV was determined by indirect immunofluorescence assay. GTC was infected with PPRV at 37°C for 48 h. Four selected VHH antibodies were used as primary antibodies and DAPI was used to stain nuclei. After 48 h, conspicuous immunofluorescent nuclei were observed. Scale bar is 100 μm.

VHH, variable domain of heavy chain; PPRV, peste des petits ruminants virus; GTC, goat tracheal epithelial cells.

Fig. 7. (A) The results of enzyme-linked immunosorbent assay test to detect the affinity of VHH2 with five proteins (H, F, P, N, M) of PPRV. (B) The results of western blot test to detect the affinity of VHH2 with five proteins (H, F, P, N, M) of PPRV.

VHH, variable domain of heavy chain; PPRV, peste des petits ruminants virus; H, hemagglutinin protein; F, the fusion protein; P, the phosphoprotein; N, the nucleocapsid protein; M, the matrix protein.