Monoclonal antibodies target conserved S1 B epitopes across deltacoronaviruses to inhibit porcine deltacoronavirus infection

Figures

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  Fig 1. Generation, purification, and characterization of mAbs against PDCoV S1. (A) SDS-PAGE analysis of the purified mAbs. (B) IFA staining of PDCoV-infected pig kidney (LLC-PK1) cells using the generated mAbs as primary antibodies at a concentration of 1 µg/mL. Pos, sera from immunized mice. Neg, mock-infected LLC-PK1 cells. Scale bar, 10 µm. (C) Detection of the S protein in the lysates of PDCoV-infected Huh7 cells via western blot with the generated mAbs. Pos, and sera from immunized mice. (D) Dot blot analysis of mAb binding to recombinant PDCoV S1-hFc proteins under native and denatured conditions. (E) The binding of the mAbs to the recombinant PDCoV S1 protein was assessed via ELISA. The EC50 (half-maximal effective concentration) values were calculated using nonlinear regression analysis. (F) The kinetics of the binding of the mAbs to the recombinant PDCoV S1 protein were determined via BLI. The association and dissociation phases were analyzed, and the equilibrium dissociation constant (KD) values were calculated via Octet system analysis software.

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  Fig 2. Epitope mapping of the mAbs. (A) Three-dimensional structure (PDB ID: 6B7N) and schematic representation of the PDCoV S protein showing differently colored S1A, S1B, S1C, and S1D domains. (B) SDS-PAGE analysis of the domains (S1A, S1B, S1C, and S1D) of the PDCoV S1 subunit fused with the hFc tag expressed in HEK293F-based eukaryotic expression systems. (C) ELISA binding of mAbs to recombinant PDCoV S1A, S1B, S1C, and S1D protein. (D) Dot blot analysis of mAbs binding to recombinant PDCoV S1A, S1B, S1C, and S1D protein under native and denatured conditions. (E) IFA staining of HEK293T cells were transfected with plasmids expressing the S1A, S1B, S1C, or S1D domains of PDCoV, and the eight generated mAbs were used to perform IFA. Scale bar, 100 µm.

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  Fig 3. Construction of rPDCoV-ΔNS6-Fluc and evaluation of mAb neutralization activity in Huh7 cells. (A) Schematic representation of the genomes of wild-type PDCoV (PDCoV-WT) and the recombinant virus rPDCoV-ΔNS6-Fluc. (B) IFA staining of LLC-PK1 cells infected with PDCoV-WT or rPDCoV-ΔNS6-Fluc. Mock, mock-infected LLC-PK1 cells. Scale bar, 200 µm. (C) Correlation analysis between the luciferase activity of rPDCoV-ΔNS6-Fluc and the MOI. (D) Growth curves of PDCoV and rPDCoV-ΔNS6-Fluc in LLC-PK1 and Huh7 cells. (E) Determination of the neutralization activity of mAbs by rPDCoV-ΔNS6-Fluc in Huh7 cells. The virus neutralization rate was calculated by comparing the RLU values of the antibody-treated wells to those of the virus-only control wells. The IC50 was determined via nonlinear regression analysis in GraphPad Prism. (F) Validation of the neutralizing activities of mAbs by wild-type PDCoV in Huh7 cells. The virus neutralization rate was quantified by IFA staining, and IC50 values were determined via nonlinear regression analysis in GraphPad Prism.

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  Fig 4. Mechanistic characterization of the S1B-specific neutralizing mAbs. (A) BLI-based assessment of APN receptor binding inhibition. (B) Antibody binding competition analyzed by BLI. (C) Neutralization assay evaluating the effect of combining mAbs 3E9 and 13D5. (D) Structural models of the antibody-RBD complexes generated using ABodyBuilder2 and HDOCK. The PDCoV RBD is shown in cyan, and pAPN is in purple according to the published PDCoV RBD-pAPN complex structure (PDB: 7VPP). Pink residues indicate previously reported pAPN-contact residues on the RBD, and orange residues indicate the predicted antibody-contact residues on the RBD. RBD residues predicted to be shared by the antibody-binding interface and the pAPN-binding interface are highlighted in red. The lower panel summarizes the overlap between the predicted antibody-contact residues and the reported pAPN-contact residues on the RBD.

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  Fig 5. Cross-reactivity and binding affinity analysis of mAbs against the S1B domains of multiple deltacoronaviruses. (A) Amino acid sequence alignment of the S1B domain from PDCoV, Haiti-Human-0256-1-2015, Haiti-Human-0081-4-2014, ISU42824, ISU73347, HKU13-3514, HKU11-934, HKU30, and HKU17. (B) IFA staining of HEK293T cells transfected with plasmids encoding the S1B domains of different deltacoronaviruses with HA tags. Scale bar, 100 µm. (C) ELISA-based determination of mAb binding to different deltacoronaviruses S1B domains. The EC50 values are shown. l.b. indicates low binding, and n.b. indicates no binding.

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  Fig 6. Evaluation of the hemagglutination inhibition activities of S1A-specific mAbs. (A) Identification of PDCoV hemagglutination activity via sialic acid binding. Neuraminidase-treated human erythrocytes were used to confirm that PDCoV-induced hemagglutination is dependent on sialic acid residues on the erythrocyte surface. (B) Determination of the hemagglutination titer of 25 μL PDCoV using human erythrocytes. (C) Hemagglutination inhibition assay evaluating the inhibitory effects of mAbs on PDCoV-induced hemagglutination.

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  Fig 7. Protective activity of mAbs in PDCoV-challenged embryonated chicken eggs. (A) Experimental design for the evaluation of the protective activity of mAbs. Created in BioRender (Z. Jiaru, 2026, https://BioRender.com/ukhax0d). (B) Survival curves of mAb-treated, DMEM-mock-treated, and blank control groups. ECE survival was recorded daily for up to 5 days post-infection. (C) Viral load in embryonic tissues.