Expression and Purification of a PEDV-Neutralizing Antibody and Its Functional Verification

Abstract

Porcine epidemic diarrhea virus (PEDV) is a highly infectious and pathogenic virus causing high morbidity and mortality, especially in newborn piglets. There remain problems with contemporary PEDV vaccines, in part because of the rapid variation of PEDV, poor conferred immunity, and numerous side effects. The ability to produce PEDV-neutralizing antibodies suggests that we may be able to increase the success rate of PEDV prevention in piglets using these antibodies. In this study, we produced an anti-PEDV S protein monoclonal antibody (anti-PEDV mAb-2) that neutralized PEDV-CV777 (a G1 strain), PEDV-SDSX16 and PEDV-Aj1102 (two G2 strains). In vivo challenge experiments demonstrated that anti-PEDV mAb-2 inhibited the PEDV infection in piglets. We also produced three HEK293 cell lines that expressed anti-PEDV mAb-2. Overall, our study showed that anti-PEDV mAb-2 produced from hybridoma supernatants effectively inhibited PEDV infection in piglets, and the recombinant HEK293 cell lines expressed anti-PEDV mAb-2 genes.

Keywords: PEDV; eukaryotic expression vector; neutralizing antibody; oral administration.

Figure 1

The vector map of eukaryotic expression vectors. (A) pCI-anti-porcine epidemic diarrhea virus (PEDV)-VL mAb. (B) pCI-anti-PEDV-VH mAb.

Figure 2

The design for in-vivo test distribution map. (A) The distribution of piglets in group A and group B. (B) The distribution of piglets in group A, group B, group C and group D. The combination of letters and numbers in the figure represents the piglet number in each group.

Figure 3

Measurement of anti-PEDV mAb-2. (A) Enzyme-linked immunosorbent assay (ELISA) measurement of serum IgG titers of the four immunized mice. (B) ELISA measurement of IgG in the supernatants of 23 hybridoma clones, the serum of mouse-3 was used as a positive control. The numbers of positive hybridoma cells were 1, 2, 15, 18 and 21. Their ODs450 were 1.462, 1.532, 0.914, 0.326 and 0.162 respectively.

Figure 4

The neutralization of PEDV by anti-PEDV mAb-2 from hybridoma supernatant and mouse ascites. (A–C) Neutralization of PEDV-CV777, PEDV-SDSX16 and PEDV-Aj1102 by anti-PEDV mAb-2 hybridoma supernatant. (D–F) Neutralization of PEDV-CV777, PEDV-SDSX16 and PEDV-Aj1102 by anti-PEDV mAb-2 mouse ascites. (G) Morphology of Vero cells after incubation for 72 h with anti-PEDV mAb-2 and PEDV. (H) Morphology of Vero cells after incubation for 72 h with PEDV only. The black arrow indicates the presence of syncytial bodies. Each column represents the average of triplicates, and each error bar indicates the standard deviations.

Figure 5

Construction of eukaryotic expression vectors containing PEDV neutralizing antibody genes. (A) pCI-Neo-hTERT digested with Nhe I and Sal I, and pCI-Neo-hTERT uncut. Empty vector is 5.5 kb. (B) Nhe I and Sal I digestion of pCI-anti-PEDV-VL and pCI-anti-PEDV-VH., VL-Fc and VH-Fc are 2 kb. (C,D) Colony PCR products from cells transfected with pCI-anti-PEDV-VL and pCI-anti-PEDV-VH. VL-Fc is 844 bp and VH-Fc is 771 bp.

Figure 6

HEK293 cells expressing anti-PEDV mAb-2 genes. (A) PCR identification of HEK293 cells expressing VL-Fc at 548 bp and VH-Fc at 410 bp from clones 5, 18, 21, 22, 24, 26, and 31. pCI-anti-PEDV-VL mAb-4 and pCI-anti-PEDV-VH mAb-12 were positive controls. (B–J) Neutralization of PEDV-CV777, PEDV-SDSX16 and PEDV-Aj1102 by supernatants from clones 24, 26, and 31.

Figure 6

HEK293 cells expressing anti-PEDV mAb-2 genes. (A) PCR identification of HEK293 cells expressing VL-Fc at 548 bp and VH-Fc at 410 bp from clones 5, 18, 21, 22, 24, 26, and 31. pCI-anti-PEDV-VL mAb-4 and pCI-anti-PEDV-VH mAb-12 were positive controls. (B–J) Neutralization of PEDV-CV777, PEDV-SDSX16 and PEDV-Aj1102 by supernatants from clones 24, 26, and 31.

Figure 7

PEDV challenge. The feces, overall anatomical changes, and histopatholog of piglets. (A–D), group A piglets. (E–H), group B piglets. Black arrow indicates the intestinal villous epithelium and villous epithelial cells (H).

Figure 8

Oral administration of anti-PEDV mAb-2 inhibits infection in piglets. (A–C) Symptom scores for diarrhea, appetite and mental state respectively. (D) Percent survival of the tested piglets.

Figure 9

Neutralization of PEDV-CV777 (A), PEDV-SDSX16 (B), and PEDV-Aj1102 (C) by serum from piglets in groups A and B. Results are expressed as the mean values from triplicate wells. Data are shown as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001).