A broadly cross-reactive antibody neutralizes and protects against sarbecovirus challenge in mice

Abstract

Severe acute respiratory syndrome coronaviruses 1 (SARS-CoV) and 2 (SARS-CoV-2), including SARS-CoV-2 variants of concern, can cause deadly infections. The mortality associated with sarbecovirus infection underscores the importance of developing broadly effective countermeasures against them, which could be key in the prevention and mitigation of current and future zoonotic events. Here, we demonstrate the neutralization of SARS-CoV; bat coronaviruses WIV-1 and RsSHC014; and SARS-CoV-2 variants D614G, B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, B.1.617.1, and B.1.617.2 by a receptor binding domain (RBD)–specific human antibody, DH1047. Prophylactic and therapeutic treatment with DH1047 was protective against SARS-CoV, WIV-1, RsSHC014, and SARS-CoV-2 B.1.351 infection in mice. Binding and structural analysis showed high affinity binding of DH1047 to an epitope that is highly conserved among sarbecoviruses. Thus, DH1047 is a broadly protective antibody that can prevent infection and mitigate outbreaks caused by SARS-related strains and SARS-CoV-2 variants. Our results also suggest that the conserved RBD epitope bound by DH1047 is a rational target for a universal sarbecovirus vaccine.

Figure 1.. The identification of broadly neutralizing antibodies.

(A) The genetic relationships of ACE2 and non-ACE2 using sarbecovirus receptor binding domains is shown. SARS-CoV-2 2AA MA is shown in purple, SARS-CoV is shown in orange, WIV-1 is shown in pink, and RsSHC014 is shown in green. The scale bar indicates the genetic distance scale among sarbecoviruses of 25%. The neutralization activity against sarbecoviruses is shown for DH1235 (B), DH1073 (C), DH1046 (D), and DH1047 (E). Data are representative of two technical replicates.

Figure 2.. The binding breadth and structural determinants of broad neutralization.

(A to D) The binding activity of cross-reactive antibodies was measured against SARS-CoV spike protein, SARS-CoV-2 spike protein, SARS-CoV-2 RBD, Pangolin GXP4L spike protein, RaTG13 spike protein, and RsSHC014 spike protein. The antibodies tested were DH1235 (A), DH1073 (B), DH1046 (C), and DH1047 (D). A cryo-EM reconstruction of DH1047 Fab bound to SARS-CoV spike protein shown (E). Spike protein is shown in gray, with the underlying fitted model shown in cartoon representation. DH1047 is colored green and the RBD it is bound to is colored black, with the receptor binding motif within the RBD colored purple. An overlay of DH1047 bound to SARS-CoV-1 and SARS-CoV-2 (PDB ID: 7LDI) spike proteins is shown in the middle panel. Overlay was performed with the respective RBDs. DH1047 bound to SARS-CoV and SARS-CoV-2 spike protein are shown in green and salmon, respectively. ACE2 (yellow surface representation, PDB 6VW1) binding to RBD is sterically hindered by DH1047. The views in panels B and C are related by a 180° rotation about the vertical axis. (F) Sequence conservation within the DH1047 HCDR3 and LCDR3s is shown among 23 sarbecoviruses.

Figure 3:. Prevention and therapy of DH1047 against SARS-CoV in aged mice.

(A) SARS-CoV mouse-adapted 15 (MA15) lung viral replication is shown. Mice (n=5 per group) treated 12 hours before infection with a control influenza mAb CH65 or one of the four broadly neutralizing antibodies DH1235, DH1073, DH1046, DH1047. Days post infection, dpi, limit of detection LoD; plaque forming units, PFU. (B) The percentage of starting weight is shown for mice (n=10 per group) treated prophylactically (12 hours before infection) or therapeutically (12 hours after infection) with DH1047 or control and challenged with SARS-CoV MA15. Data are presented as mean±SEM. (C) Lung viral replication of SARS-CoV MA15 is shown for mice treated prophylactically or therapeutically with DH1047 and control at 4 days post infection. (D) Macroscopic lung discoloration scores are shown for mice treated with DH1047 or control antibody prophylactically and therapeutically. (E) Lung pathology at day 4 post infection was measured by acute lung injury (ALI) scores in mice treated with DH1047 or control prophylactically and therapeutically. (F) Lung pathology at day 4 post infection was measured by diffuse alveolar damage (DAD) in mice treated prophylactically and therapeutically with DH1047 and control. (G) Pulmonary function was measured by whole body plethysmography in mice treated with DH1047 or control mAb prophylactically or therapeutically. (H) Percent survival is shown for mice treated with control or DH1047 prophylactically or therapeutically and challenged with SARS-CoV MA15. Horizontal bars in (A) and (C to G) indicate mean. P-values shown in (B and G) were calculated using a two-way ANOVA followed by Tukey’s multiple comparisons test. P-values are for (A) and (C to E) were calculated using a one-way ANOVA followed by Dunnett’s multiple comparisons. P values for (H) were calculated using a Mantel-Cox test. ^**p<0.01; ^***p<0.001; ^****p<0.0001.

Figure 4:. Prophylactic and therapeutic activity of DH1047 against SARS-related bat coronaviruses and the in vitro neutralization against the SARS-CoV-2 variants.

(A) Lung viral replication of WIV-1 is shown for mice (n=4 to 5 per group) treated prophylactically or therapeutically with DH1047 or control at 2 days post infection. Horizontal bars indicate mean. (B) Lung viral replication of RsSHC014 is shown for mice (n=5 to 10 per group) treated prophylactically or therapeutically with DH1047 or control at 2 days post infection. Horizontal bars indicate mean. (C) Live virus neutralization of SARS-CoV-2 D614G, B.1.1.7, and B.1.351 variants by DH1047 is shown. Data are representative of two technical replicates. (D) The comparison of the DH1047 neutralization activity against SARS-CoV-2 variants is shown for pseudovirus and live virus neutralization assays. NT, not tested. P-values in (A) and (B) were calculated using a one-way ANOVA followed by Dunnett’s multiple comparisons test. *p<0.05; ^**p<0.01.

Figure 5:. Prophylaxis and therapy of DH1047 against SARS-CoV-2 B.1.351 in mice.

(A) Percent of starting weight is shown for mice (n=10 per group) treated prophylactically (12 hours before infection) or therapeutically (12 hours after infection) with DH1047 or control antibody and challenged with SARS-CoV-2 B.1.351. (B) Lung viral replication is shown at 4 days post infection for mice infected with SARS-CoV-2 B.1.351 and treated prophylactically or therapeutically with DH1047 or control. (C) Macroscopic lung discoloration scores are shown at 4 days post infection for mice infected with SARS-CoV-2 B.1.351 and treated prophylactically or therapeutically with DH1047 or control. (D) Lung pathology measured by acute lung injury (ALI) scores is shown at 4 days post infection for mice infected with SARS-CoV-2 B.1.351 and treated prophylactically or therapeutically with DH1047 or control. (E) Lung pathology measured by diffuse alveolar damage (DAD) is shown at 4 days post infection for mice infected with SARS-CoV-2 B.1.351 and treated prophylactically or therapeutically with DH1047 or control. (F) Percent survival is shown for SARS-CoV-2 B.1.351-infected mice treated with DH1047 prophylactically or therapeutically with DH1047 or control. (G) Percent of starting weight is shown for mice (n=10 per group) treated prophylactically with DH1047 (12 hours before infection) at de-escalating doses of 10, 5, and 1mg/kg or with 10mg/kg control mAb, CH065. (H) Lung viral titers in control and DH1047-treated mice at 10, 5, and 1mg/kg. (I) Lung discoloration in control and DH1047-treated mice at the various mAb doses. Data in (A and G) are presented as mean±SEM. Horizontal bars in (B to E) and (H and I) represent mean. P-values shown in (A and G) are from a two-way ANOVA followed by Tukey’s multiple comparisons test, and P-values shown in (B to E) and (H and I) are from a one-way ANOVA followed by Dunnett’s multiple comparisons test. Differences in survival were measured by a Mantel-Cox test. ^**p<0.01; ^***p<0.001; ^****p<0.001; ns, not significant. (J) DH1047 binding is shown relative to binding of other known antibody classes that bind the RBD. RBD is shown in black with the ACE2 footprint on the RBD colored yellow. DH1047 is shown in cartoon representation and colored green. The other antibodies are shown as transparent surfaces: C105 (pale cyan, Class 1, PDB ID: 6XCN and 6XCA), DH1041 (light blue, Class 2, PDB ID: 7LAA), S309 (wheat, Class 3, PDB ID:6WS6 and 6WPT) and CR3022 (pink, Class 4, PDB ID: 6YLA)