Broadly neutralizing antibodies against emerging delta-coronaviruses

Porcine deltacoronavirus (PDCoV) spillovers were recently detected in children with acute undifferentiated febrile illness, underscoring recurrent zoonoses of divergent coronaviruses. To date, no vaccines or specific therapeutics are approved for use in humans against PDCoV. To prepare for possible future PDCoV epidemics, we isolated human spike (S)-directed monoclonal antibodies from transgenic mice and found that two of them, designated PD33 and PD41, broadly neutralized a panel of PDCoV variants. Cryo-electron microscopy structures of PD33 and PD41 in complex with the PDCoV receptor-binding domain and S ectodomain trimer provide a blueprint of the epitopes recognized by these mAbs, rationalizing their broad inhibitory activity. We show that both mAbs inhibit PDCoV by competitively interfering with host APN binding to the PDCoV receptor-binding loops, explaining the mechanism of viral neutralization. PD33 and PD41 are candidates for clinical advancement, which could be stockpiled to prepare for possible future PDCoV outbreaks.

Table S1.Table of the KDs, association (Ka) and dissociation rate constants (Kdis) for duplicate lots of PDCoV RBDIL121_2014 and Fab fragments of PD33 and PD41.
Table S2.Table of the sequence information of PDCoV S variants as full length, ectodomain and RBD along with hAPN and gAPN sequences as full length and ectodomain constructs.A, List of PDCoV variants used in the study and the genebank ID associated for those variants.The table includes information for the APN constructs used in the study and the genebank ID corresponding to those constructs.B, List of the PDCoV variants tested in the study and the RBD mutations found in these variants relative to PDCoVIL121_2014.Table S3.CryoEM data collection and refinement statistics.

Figure S1 .
Figure S1.Screening for binding and neutralization of a panel of human mAbs (PD3, PD8, PD17, PD20, PD23, PD24, PD25, PD26, PD27, PD28, PD30, PD31, PD32, PD33, PD35, PD36, PD37, PD41) isolated from transgenic mice.A, Screening of isolated mAbs for binding to PDCoV RBD immobilized at the surface of BLI biosensors.B, Screening of isolated mAbs immobilized at the surface of BLI biosensors for binding to PDCoV S C, Evaluation of binding of the isolated mAbs to PDCoV S measured by ELISA.Each point represents the mean of technical quadruplets.Standard deviations shown as error bars.D, Dose-dependent mAb-mediated neutralization of PDCoV S VSV using HEK293T target cells transiently transfected with galline APN (gAPN).Each point represents the mean of technical triplicates.Standard deviations shown as error bars.

Figure S2 :
Figure S2: Kinetics of PD33 and PD41 Fabs binding to PDCoV RBD.BLI analysis of PD33 and PD41 Fab fragments binding to immobilized PDCoVIL121_2014 RBD immobilized onto Ni-NTA tips.Fits to the data are shown as black dotted lines and were used to determine the binding affinity (KD) of the PD33 and PD41 Fab fragments to PDCoV RBD.Two biological replicates are shown for each Fab.

Figure S3 .
Figure S3.Assessment of PD33 and PD41 neutralization breadth.Dose-dependent mAb-mediated neutralization of PDCoV S VSV variants using HEK293T target cells transiently transfected with human APN.A-C are three independent runs with 3 different batches of pseudovirus.

Figure S4 :
Figure S4: Cryo-EM data collection and refinement of PDCoV RBDIL2014 bound to the PD33 Fab fragment.A, Representative electron micrograph (3.0 µm defocus, scale bar = 100 nm) B, 2D class averages (scale bar = 150 Å).C, Gold-standard Fourier shell correlation curve for the cryoEM reconstruction.The 0.143 cutoff is indicated with a gray dashed line.D, 3D reconstruction of PDCoV RBD bound to PD33 colored by local resolution calculated using CryoSPARC.E, Flow chart of the pipeline for processing.CTF: contrast transfer function; NUR: non-uniform refinement.

Figure S5 .
Figure S5.Functional characterization of PDCoV S variants.A, Entry of PDCoV S VSV variants into HEK293T target cells transiently transfected with human APN.B, Western blot quantification of PDCoV S incorporation in VSV pseudotypes for each of the three biological replicates used in panel A. C-D, BLI analysis of hAPN-Fc binding to the PDCoVIL121_2014 RBD (left) or to the PDCoVSD_2018 (right) immobilized onto Ni-NTA tips.Fits to the data are shown as black dotted lines and were used to determine the apparent binding affinity (KD,app) of hAPN-Fc fragments to the two PDCoV RBDs.

Figure S6 :
Figure S6: Cryo-EM data collection and refinement of PDCoV RBDSD2018 bound to the PD41 Fab fragment.A, Representative electron micrograph (2.5 µm defocus, scale bar = 100 nm) B, 2D class averages (scale bar = 150 Å).C, Gold-standard Fourier shell correlation curve for the cryoEM reconstruction.The 0.143 cutoff is indicated with a black dashed line.D, 3D reconstruction of PDCoV RBD bound to PD41 colored by local resolution calculated using CryoSPARC.E, Flow chart of the pipeline for processing.CTF: contrast transfer function; NUR: non-uniform refinement.

Figure S7 :
Figure S7: Superimposition of the PD33-bound PDCoV RBD and of the PD41-bound PDCoV RBDSD2018 structures showing recognition of distinct but overlapping epitopes.A, PD33 (purple and magenta surfaces for the heavy and light chains, respectively) and PD41 (green and orange surfaces for the heavy and light chains, respectively) recognize overlapping epitopes on the PDCoV RBD (cyan ribbon).N-linked glycans are rendered as blue surfaces.The red star indicates steric clashes.B, BLI analysis of Fab PD33 binding to the PDCoV RBD immobilized on biosensors in the presence and absence of Fab PD41.C, BLI analysis of Fab PD41 binding to the PDCoV RBD immobilized on biosensors in the presence and absence of Fab PD33.