Breadth of ACE2 receptor usage predicts host range and antigenic relatedness across bat sarbecoviruses

Figures

• 

  Fig 1. Phylogenetic and geographic distribution of bat sarbecoviruses and their bat ACE2 tropism. (A) Evolutionary history of bat sarbecovirus RBDs (NCBI reference shown for 124 amino acid sequences) was inferred by using the Maximum Likelihood method and Whelan and Goldman model. Initial tree(s) for the heuristic search were obtained automatically by Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances (225 total positions) estimated using the JTT model with discrete Gamma distribution (5 categories (+G, parameter = 0.3524)), and then selecting the topology with superior log-likelihood value. Evolutionary analyses were conducted in MEGA 11. Bat sarbecovirus RBDs cluster into clades: clade Ia (yellow), clade Ib (orange), clade II (blue), clade III (green), clade IV (purple) and clade V (red). Viruses selected for analysis in this study are highlighted in red. (B) World map highlighting the geographic location where sarbecoviruses investigated in this study were first isolated (coloured in blue), along with the distribution of the bat species these were isolated from (https://www.iucnredlist.org/). (C) Structural images showing the binding of Rhinolophus affinis ACE2…

• 

  Fig 2. Cryo-EM structure of RhGB07 trimeric spike in a 3-RBD down state. (A) Left: CryoEM density of the trimer spike coloured by the protomer chains the density originates from. Glycans are highlighted in teal. Centre: the trimeric spike viewed from the top rendered in cartoon representation. Right: the isolated RhGB07 RBD represented as cartoon in grey with two glycans of interest shown. (B) The C-alpha trace of the RBD of RhGB07 rendered as balls-and-sticks superimposed with the RBD of SARS-CoV-1 (PDB 7DF3) and SARS-CoV-2 (PDB 5X5B). The insets show a zoomed-in view of two regions of the RBD that show the greatest changes in the position of the C-alpha of the RBD. The region containing the ins1-loop and delta4-loop coincides with putative ACE2-interacting residues based on mapping the equivalent residues of SARS-CoV-1 and SARS-CoV-2. The ACE2-interacting residues were determined by superimposing SARS-CoV-1 and RhGB07 RBD onto the SARS-CoV-2 RBD – ACE2 (PDB: 6M0J) structure and identifying interacting residues. Highlighted residues of RhGB07 show the putative interacting residues that differ in identity to SARS-CoV-2. (C) Schematic depicting mutants generated of RhGB07 and SARS-CoV-2…

• 

  Fig 3. Bat sarbecovirus usage of mammalian ACE2. (A) Heatmap showing the receptor usage of bat sarbecovirus-pseudotyped viruses with mammalian ACE2s, including human, livestock/pets, rodents, potential intermediate hosts and monkeys. The mean log RLU from 3 separate experiments is shown and a pDISPLAY control to illustrate the background levels of entry. (B) Surface representation of human ACE2 coloured with mammalian ACE2 conservation (human ACE2: PDB 6M0J), with the outlined region denoting the RBD-interacting sites, with individual amino acids labelled. (C) WebLogo (University of California, Berkley, USA) plots summarising the amino acid divergence within the mammalian ACE2 sequences used in this screen. The single-letter amino acid code is used with the vertical height presenting the relative frequency of that amino acid at each position. The positions at which ACE2 interacts with SARS-CoV-2 RBD are shown, N-terminal to C-terminal, with different colours representing amino acid conservation between the mammalian ACE2s examined in this study. The data underlying this Figure can be found in S1 Data.

• 

  Fig 4. Species tropism of bat and mammalian ACE with SARS-CoV-2 variants. (A) Schematic of the emergence of SARS-CoV-2 variants depicting the divergence of the B lineage variants, including those variants of concern that grew to global dominance (B.1, Alpha, Beta, Delta, Omicron [black lines]), and the Omicron subvariants [red lines], some of which emerged via recombination events (dotted lines). Heatmap showing the bat and mammalian ACE2 usage of SARS-CoV-2 variants with (B) the B.1, Alpha, Beta, Delta and BA.1 variants, or (E) BA.2 and its subvariants BA.4/5, XBB, XBB1.5, BA.2.86. Data are shown as the mean fold change in pseudotype entry (RLU) of 3 separate experiments, relative to human ACE2. (C) Structure of SARS-CoV-2 BA.1 (PDB 7XO7) with mutations listed compared to B.1. Zoomed images of the Spike protein for B.1 (PDB 6M0J) and BA.1 (PDB 7WBP), highlighting key residues at positions 417, 484, 493 and 501 and the resultant interactions with human ACE2. (D) Specific amino acids within the B.1 RBD were mutagenised to make the RBD more BA.1-like (K417N, E484A, Q493R or N501Y) (top panel), or within BA.1 RBD to make the RBD more B.1-like (N417K, A484E, R493Q or Y501N) (bottom panel),…

• 

  Fig 5. Antigenicity of bat sarbecoviruses using SARS-CoV-2 polyclonal convalescent sera. (A) Table outlining the different convalescent plasma/serum used to investigate the antigenicity of bat sarbecoviruses. These include samples from individuals following infection with the wildtype (WT), Alpha, Gamma, Delta, or Omicron BA.1 variants, either confirmed by PCR or assumed based on epidemiological data. The sample set also includes WHO International Standards provided by MRHA (NIBSC), pooled vaccinee plasma (low and high titre) and negative plasma, pooled from individuals pre-2019. Information about the COVID-19 standards and reagents can be found here: WHO/BS/2022.2427: Establishment of the 2nd WHO International Standard for anti-SARS-CoV-2 immunoglobulin and Reference Panel for antibodies to SARS-CoV-2 variants of concern. (B) Heatmap showing the log (IC50) values of neutralisation (the dilution at which there was a 50% reduction in luciferase activity) of SARS-CoV-2, BANAL-20-52, BANAL-20-103, BANAL-20-236, Rs4231, WIV-1, RhGB07, Rc-o319 and BA.1 viruses in the presence of the MHRA (NIBSC) reference reagents. HEK293T cells stably expressing human ACE2 were used for all neutralisation…

• 

  Fig 6. Binding of monoclonal antibodies isolated from SARS-CoV-2 breakthrough infections to bat sarbecovirus RBDs. (A) Neutralisation of SARS-CoV-2 and Rs4231 RBD chimeras with SARS-CoV-2 RBD-specific monoclonal antibodies (mAb) (IC50 µg/ml), with dotted lines linking matched samples. The p values are shown, calculated by comparing the change in mean IC50 value and by using Tukey’s multiple comparison tests, respectively. (B) Spike structure of SARS-CoV-2 with 2 RBD-down and 1 RBD-up (PDB 7QTI) with the binding epitope regions of the different mAb competition groups is shown with both a side- and top-view (Group 1 = purple, Group 2 = orange, Group 3 = red, Group 3.5 = blue, Group 4 = green, Group 8 = non-RBD, NTD-specific, defined previously [46–48]. (C) SARS-CoV-2 RBD structures (PDB 7QTI) highlighting a broad epitope footprint of the mAb competition groups corresponding binding data. Each of the plots shows a single mAb binding in an ELISA using purified (C) SARS-CoV-2 or (E) other bat sarbecovirus RBDs. Plots show raw optical density (OD) values with curves calculated using a least-squares regression non-linear fit. (D) Epitope binning by surface plasmon resonance (SPR) identifies 5…

• 

  Fig 7. Antigenicity of bat sarbecoviruses using monoclonal antibodies isolated from SARS-CoV-2 breakthrough infections. (A) Dissociation constant (KD (M), 2−4 replicates + sd) of mAb binding kinetics calculated by SPR (bars) and ELISA EC50 (µg/ml, 2 replicates + sd) values (broken lines with filled symbols) for a subset of mAbs and sarbecovirus RBDs. For KD, no bar indicates no binding by SPR. For EC50, lower limit of detection: <0.08 µg/ml, upper limit of detection: >5 µg/ml. (B) Neutralisation (IC50) titres shown for each mAb against bat sarbecovirus pseudotyped viruses (µg/ml), with limits of detection indicated (lower limit: <0.08 µg/ml; upper limit: >50 µg/ml for IC50). HEK293T cells stably expressing human ACE2 were used for all neutralisation assays. apart from RhGB07 and Rc-o319, where HEK293T cells overexpressing Rh. alcyone or Rh. cornutus were used, respectively. Scale bars (right) represent direction of increased binding or neutralisation. (C) RhGB07 trimer and RBD, with antibody competition groups highlighted, and are coloured with the same colouring as the SARS-CoV-2 trimer in (Fig 6B). Regions of divergence in binding epitopes in RhGB07 compared to SARS-CoV-2 are…