mammalian ace2 constructs (Bio Basic Canada)

Bio Basic Canada mammalian ace2 constructs

(A) BHK-21 cells were transfected with HA-tagged bat <t>ACE2-expressing</t> constructs and baited with His tagged-purified RBD proteins for bat sarbecoviruses. Cells were stained with anti-HA PE conjugated antibody (ACE2) and anti-His APC conjugated antibody (RBD). Live and singlet BHK-21 cells were gated as PE+ APC+ (ACE2+RBD+), relative to mock-transfected cells (pDISPLAY). (B) The SE% Dymax metric was used to calculate the percentage of ACE2+RBD+ cells, shown for all RBDs tested with our library of bat ACE2 and human ACE2. Representative datasets are shown for mock-transfected cells and raccoon dog ACE2 with SARS-CoV-2 RBD, with their respective SE Dymax % positive value noted. (C) Heatmap showing the binding of purified bat sarbecovirus proteins to ACE2s of human, M. lucifugus Rh. cornutus by ELISA (pink, EC 50 µg/ml) or flow cytometry (blue, % ACE2 + RBD + cells). Data is representative of a mean of 3 separate experiments. A cross in a box indicates this data was not obtained.

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(A) BHK-21 cells were transfected with HA-tagged bat ACE2-expressing constructs and baited with His tagged-purified RBD proteins for bat sarbecoviruses. Cells were stained with anti-HA PE conjugated antibody (ACE2) and anti-His APC conjugated antibody (RBD). Live and singlet BHK-21 cells were gated as PE+ APC+ (ACE2+RBD+), relative to mock-transfected cells (pDISPLAY). (B) The SE% Dymax metric was used to calculate the percentage of ACE2+RBD+ cells, shown for all RBDs tested with our library of bat ACE2 and human ACE2. Representative datasets are shown for mock-transfected cells and raccoon dog ACE2 with SARS-CoV-2 RBD, with their respective SE Dymax % positive value noted. (C) Heatmap showing the binding of purified bat sarbecovirus proteins to ACE2s of human, M. lucifugus Rh. cornutus by ELISA (pink, EC 50 µg/ml) or flow cytometry (blue, % ACE2 + RBD + cells). Data is representative of a mean of 3 separate experiments. A cross in a box indicates this data was not obtained.

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) BHK-21 cells were transfected with HA-tagged bat ACE2-expressing constructs and baited with His tagged-purified RBD proteins for bat sarbecoviruses. Cells were stained with anti-HA PE conjugated antibody (ACE2) and anti-His APC conjugated antibody (RBD). Live and singlet BHK-21 cells were gated as PE+ APC+ (ACE2+RBD+), relative to mock-transfected cells (pDISPLAY). (B) The SE% Dymax metric was used to calculate the percentage of ACE2+RBD+ cells, shown for all RBDs tested with our library of bat ACE2 and human ACE2. Representative datasets are shown for mock-transfected cells and raccoon dog ACE2 with SARS-CoV-2 RBD, with their respective SE Dymax % positive value noted. (C) Heatmap showing the binding of purified bat sarbecovirus proteins to ACE2s of human, M. lucifugus Rh. cornutus by ELISA (pink, EC 50 µg/ml) or flow cytometry (blue, % ACE2 + RBD + cells). Data is representative of a mean of 3 separate experiments. A cross in a box indicates this data was not obtained.

Article Snippet: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

Techniques: Transfection, Expressing, Construct, Purification, Staining, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

(A) Evolutionary history of bat sarbecovirus RBDs (NCBI reference shown for 110 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 = 0.3460)), and then selecting the topology with superior log likelihood value. Evolutionary analyses were conducted in MEGA 11. Of note, the HKU3-7 and HKU3-8, and the HKU3-1, 2, 3, 4, 5, 6, 7, 9, 19, 11 and 12 Spike sequences are identical, so the HKU3-8 and HKU3-1 sequences were used as representatives, respectively. 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 with SARS-CoV-2 RBD (PDB 7XA7). Offset images highlight the conserved amino acid residues across the different bat ACE2s relative to Rh. affinis ACE2, and the conservation of bat sarbecovirus RBDs compared to SARS-CoV-2, screened in this study. The ACE2-RBD interacting residues are denoted. (D) Maximum likelihood phylogeny of the bat sarbecoviruses full length Spike and bat ACE2 amino acid sequences are shown. Bat sarbecovirus Spikes were used to generate lentiviral-based pseudotypes and used to infect BHK-21 cells overexpressing different bat ACE2s or human ACE2. A heatmap illustrating receptor usage is shown, representing the mean log RLU of 3-5 separate experiments. A vector only control (pDISPLAY) was included to demonstrate specificity and to set the background signal. The cognate bat receptor for each virus, where possible is highlighted with a red box. Absence of a red square is indicative of the lack of cognate receptor from our screen, either due to unavailability of ACE2 sequence, or because the original bat host is yet unknown (RhGB07: Rh. hipposideros ; SARS-CoV-2, SARS-CoV-1: unknown; BANAL-20-52: Rh. malayanus ; BANAL-20-236: Rh. marshalli ; RacCS203 – Rh. acuminatus ). (E) Sequence alignment of bat sarbecovirus RBDs at the ACE2-binding interface, with key amino acid residues denoted, using SARS-CoV-2 numbering. Regions of amino acid deletions between sequences are denoted in red. The corresponding human and Rh.affinis ACE2 residues that bind SARS-CoV-2 RBD are highlighted, with black indicating amino acid residues that are the same between human ACE2 and Rh. affinis ACE2, green indicating the same residues but a different amino acid, and the remainder highlighting unique binding residues for human ACE2 (blue) or Rh. affinis ACE2 (pink) only.

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) Evolutionary history of bat sarbecovirus RBDs (NCBI reference shown for 110 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 = 0.3460)), and then selecting the topology with superior log likelihood value. Evolutionary analyses were conducted in MEGA 11. Of note, the HKU3-7 and HKU3-8, and the HKU3-1, 2, 3, 4, 5, 6, 7, 9, 19, 11 and 12 Spike sequences are identical, so the HKU3-8 and HKU3-1 sequences were used as representatives, respectively. 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 with SARS-CoV-2 RBD (PDB 7XA7). Offset images highlight the conserved amino acid residues across the different bat ACE2s relative to Rh. affinis ACE2, and the conservation of bat sarbecovirus RBDs compared to SARS-CoV-2, screened in this study. The ACE2-RBD interacting residues are denoted. (D) Maximum likelihood phylogeny of the bat sarbecoviruses full length Spike and bat ACE2 amino acid sequences are shown. Bat sarbecovirus Spikes were used to generate lentiviral-based pseudotypes and used to infect BHK-21 cells overexpressing different bat ACE2s or human ACE2. A heatmap illustrating receptor usage is shown, representing the mean log RLU of 3-5 separate experiments. A vector only control (pDISPLAY) was included to demonstrate specificity and to set the background signal. The cognate bat receptor for each virus, where possible is highlighted with a red box. Absence of a red square is indicative of the lack of cognate receptor from our screen, either due to unavailability of ACE2 sequence, or because the original bat host is yet unknown (RhGB07: Rh. hipposideros ; SARS-CoV-2, SARS-CoV-1: unknown; BANAL-20-52: Rh. malayanus ; BANAL-20-236: Rh. marshalli ; RacCS203 – Rh. acuminatus ). (E) Sequence alignment of bat sarbecovirus RBDs at the ACE2-binding interface, with key amino acid residues denoted, using SARS-CoV-2 numbering. Regions of amino acid deletions between sequences are denoted in red. The corresponding human and Rh.affinis ACE2 residues that bind SARS-CoV-2 RBD are highlighted, with black indicating amino acid residues that are the same between human ACE2 and Rh. affinis ACE2, green indicating the same residues but a different amino acid, and the remainder highlighting unique binding residues for human ACE2 (blue) or Rh. affinis ACE2 (pink) only.

Techniques: Isolation, Binding Assay, Plasmid Preparation, Control, Virus, Sequencing

Expression of tagged ACE2 proteins was carried out in lysates from BHK-21 cells transfected with ACE2 constructs and probed for HA expression by Western blotting. Sarbecovirus Spikes were used to generate lentivral-based pseudotypes, and the viral supernatant purified by ultra-centrifugation under a 20% sucrose cushion. Purified viruses were probed with an anti-FLAG antibody (except RhGB07 and RfGB02, which are untagged), an anti-Spike antibody (S, full-length Spike; S1/S2, cleaved variant) and the lentivirus internal control, anti-p24 antibody.

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: Expression of tagged ACE2 proteins was carried out in lysates from BHK-21 cells transfected with ACE2 constructs and probed for HA expression by Western blotting. Sarbecovirus Spikes were used to generate lentivral-based pseudotypes, and the viral supernatant purified by ultra-centrifugation under a 20% sucrose cushion. Purified viruses were probed with an anti-FLAG antibody (except RhGB07 and RfGB02, which are untagged), an anti-Spike antibody (S, full-length Spike; S1/S2, cleaved variant) and the lentivirus internal control, anti-p24 antibody.

Techniques: Expressing, Transfection, Construct, Western Blot, Purification, Centrifugation, Variant Assay, Control

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) Heatmap showing virus entry by cell-cell fusion. HEK293T cells stably expressing half of a split GFP-Renilla (RLuc-GFP 1-7) luciferase reporter were transfected with bat sarbecovirus Spikes, and co-cultured with BHK-21 cells expressing the other half of the split reporter (RLuc-GFP 8-11), transfected with bat ACE2s. Productive cell-cell fusion results in reconstitution of the split reporter, from which luciferase signals can be measured. Data is shown as log (RLU) and is the average of three separate experiments. A pDISPLAY vector control was included to set the baseline value to account for background signals. The red boxes indicate the predicted cognate bat receptor for each bat sarbecovirus, where known. (B) XY scatter plots for each of the viruses assessed for their receptor usage profile, correlating the log (RLU) of pseudoparticle entry (X) against cell-cell fusion entry (Y), excluding clade II viruses that do not use ACE2 as their entry receptor (RfGB02: r= 0.8244, p<0.0001; RhGB07: r=0.9335, p<0.0001; Rc-o319: r=0.9467, p<0.0001; SARS-CoV-2: r = 0.6684, p=0.0024; BANAL-20-103: r=0.5308, p=0.0234; BANAL-20-236: r=0.6096, p=0.0072; BANAL-20-52: r=0.7343, p=0.0005; RaTG13: r=0.7719, p=0.0002; SARS-CoV-1: r=0.9005, p<0.0001; WIV-1: r=0.7724, p=0.0002; Rs4231: r=0.8259, p<0.0001). Each data point is labelled with the ACE2 corresponding ACE2 receptor and a Pearson’s correlation calculated with 95% confidence intervals. The Pearson r value and p value are both documented.

Techniques: Virus, Stable Transfection, Expressing, Luciferase, Transfection, Cell Culture, Plasmid Preparation, Control

(A) left: CryoEM density of the trimer spike coloured by the protomer chains the density originates from. Glycans are highlighted in teal. Center: 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 and SARS-CoV-2. 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.

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) left: CryoEM density of the trimer spike coloured by the protomer chains the density originates from. Glycans are highlighted in teal. Center: 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 and SARS-CoV-2. 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.

Techniques: Isolation

(A) Sequence conservation of selected sarbecoviruses were mapped on to the RhGB07 spike. Areas least conserved are in red and most conserved are blue. The least conserved regions are at the top of the RBD, the NTD and close to a putative polybasic site. (B) The C-alpha trace of the RBD of RhGB07 rendered as balls-and-sticks superimposed with the RBD of PRD-0038 apo or PRD-0039 bound to R. Alcyone ACE2. 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.

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) Sequence conservation of selected sarbecoviruses were mapped on to the RhGB07 spike. Areas least conserved are in red and most conserved are blue. The least conserved regions are at the top of the RBD, the NTD and close to a putative polybasic site. (B) The C-alpha trace of the RBD of RhGB07 rendered as balls-and-sticks superimposed with the RBD of PRD-0038 apo or PRD-0039 bound to R. Alcyone ACE2. 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.

Techniques: Sequencing

(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.

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (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.

Techniques: Control

(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), either by themselves or in combination. Entry of these mutants was tested with Rh. cornutus, Rh. pusillus , Rh. ferrumequinum , and L. Lyra ACE2s. Data is shown as log fold-change in pseudotype entry, with green bars indicating an increase in virus entry and blue bars a decrease in entry, relative to B.1 (top panel) or BA.1 (bottom panel), highlighted also in .

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (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), either by themselves or in combination. Entry of these mutants was tested with Rh. cornutus, Rh. pusillus , Rh. ferrumequinum , and L. Lyra ACE2s. Data is shown as log fold-change in pseudotype entry, with green bars indicating an increase in virus entry and blue bars a decrease in entry, relative to B.1 (top panel) or BA.1 (bottom panel), highlighted also in .

Techniques: Virus

(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 the 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 (IC 50 ) 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 assays, apart from RhGB07 and Rc-o319, where HEK293T cells overexpressing Rh. alcyone or Rh. cornutus were used, respectively (indicated by an asterisk). (C) Violin plots show collated IC 50 values for the neutralisation of these viruses with each sera set, with the median, upper and lower quartiles (red lines) and limit of detection (black dotted line) shown. A one-way ANOVA with Dunnet’s multiple comparison was performed, comparing to SARS-CoV-2, with significance values indicated (* = p<0.05, ** = p<0.005, ***= p<0.0005, ns = non-significant).

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (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 the 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 (IC 50 ) 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 assays, apart from RhGB07 and Rc-o319, where HEK293T cells overexpressing Rh. alcyone or Rh. cornutus were used, respectively (indicated by an asterisk). (C) Violin plots show collated IC 50 values for the neutralisation of these viruses with each sera set, with the median, upper and lower quartiles (red lines) and limit of detection (black dotted line) shown. A one-way ANOVA with Dunnet’s multiple comparison was performed, comparing to SARS-CoV-2, with significance values indicated (* = p<0.05, ** = p<0.005, ***= p<0.0005, ns = non-significant).

Techniques: Clinical Proteomics, Infection, Luciferase, Activity Assay, Stable Transfection, Expressing, Comparison

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) Infection of HEK293T cells stably expressing hACE2 with SARS-CoV-2, Rs4231 or chimeric pseudoparticles (Rs4231 + SARS-CoV-2 RBD, SARS-CoV-2 + Rs4231 RBD) Virus was titrated 5-fold on cells and Firefly luciferase signals measured; a negative control (non-enveloped, NE) was also included. (B) Pseudoparticle entry assay using SARS-CoV-2 and Rs4231 chimeras, on a subset of ACE2s that had striking entry profile in the initial screen (human, Rh. sinicus , Rh. shameli, Rh. macrotis, T. melanopogan, mouse, Syrian hamster, pig, raccoon dog and marmoset ACE2). The heatmap shows log(RLU) of psuedotype entry, with pDISPLAY included as a control to establish background luciferase signal.

Techniques: Infection, Stable Transfection, Expressing, Virus, Luciferase, Negative Control, Control

(A) Dissociation constant (K D (M), 2-4 replicates ± sd) of mAb binding kinetics calculated by SPR (bars) and ELISA EC 50 (µg/ml, 2 replicates ± sd) values (broken lines with filled symbols) for a subset of mAbs and sarbecovirus RBDs. For K D , no bar indicates no binding by SPR. For EC 50 , lower limit of detection: <0.08µg/ml, upper limit of detection: >5µg/ml. (B) Neutralisation (IC 50 ) titres shown for each mAb against bat sarbecovirus pseudotyped viruses (µg/ml), with limits of detection indicated (lower limit: <0.08µg/ml; upper limit: >50µg/ml for IC 50 ). 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. (C) RhGB07 trimer and RBD, with antibody competition groups highlighted, and are coloured with the same colouring as the SARS-CoV-2 trimer in . Regions of divergence in binding epitopes in RhGB07 compared to SARS-CoV-2 are depicted, with amino acid residues denotes. Colours in all graphs/structures indicate mAb binding competition groups (Group 1 = purple, Group 2 = orange, Group 3 = red, Group 3.5 = blue, Group 4 = green). EC 50 , K D and IC 50 values be found in .

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) Dissociation constant (K D (M), 2-4 replicates ± sd) of mAb binding kinetics calculated by SPR (bars) and ELISA EC 50 (µg/ml, 2 replicates ± sd) values (broken lines with filled symbols) for a subset of mAbs and sarbecovirus RBDs. For K D , no bar indicates no binding by SPR. For EC 50 , lower limit of detection: <0.08µg/ml, upper limit of detection: >5µg/ml. (B) Neutralisation (IC 50 ) titres shown for each mAb against bat sarbecovirus pseudotyped viruses (µg/ml), with limits of detection indicated (lower limit: <0.08µg/ml; upper limit: >50µg/ml for IC 50 ). 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. (C) RhGB07 trimer and RBD, with antibody competition groups highlighted, and are coloured with the same colouring as the SARS-CoV-2 trimer in . Regions of divergence in binding epitopes in RhGB07 compared to SARS-CoV-2 are depicted, with amino acid residues denotes. Colours in all graphs/structures indicate mAb binding competition groups (Group 1 = purple, Group 2 = orange, Group 3 = red, Group 3.5 = blue, Group 4 = green). EC 50 , K D and IC 50 values be found in .

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing

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