Dendritic Cell‐Mediated Cross‐Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS‐CoV‐2

SARS-CoV-2 B.1.351 and B.1.167.2 viruses used in this study were obtained through the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 653316. SARS-CoV-2 B.1 (MAD6 isolate) was kindly provided by José M. Honrubia and Luis Enjuanes (CNB-CSIC, Madrid, Spain). The authors thank Centro de Investigación en Sanidad Animal (CISA)-Instituto Nacional de Investigaciones Agrarias (INIA-CSIC) (Valdeolmos, Madrid, Spain) for the BSL-3 facilities. Research in LAV laboratory was funded by the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 y COVID19); the MCIN/AEI/10.13039/501100011033 (PID2020-117323RB-I00 and PDC2021-121711-I00), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (DTS20/00089), partially supported by the ERDF, the Spanish Association Against Cancer (AECC 19084); the CRIS Cancer Foundation (FCRISIFI-2018 and FCRIS-2021-0090), the Fundación Caixa-Health Research (HR21-00761 project IL7R_LungCan), and the Comunidad de Madrid (P2022/BMD-7225 NEXT_GEN_CART_MAD-CM). Work in the DS laboratory was funded by the CNIC; the European Union’s Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator Grant 725091; MCIN/AEI/10.13039/501100011033 (PID2019-108157RB); Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); Atresmedia (Constantes y Vitales prize); Fondo Solidario Juntos (Banco Santander); and “La Caixa” Foundation (LCF/PR/HR20/00075). The CNIC was supported by the ISCIII, the MCIN and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020- 001041-S funded by MCIN/AEI/10.13039/501100011033). Research in RD laboratory was supported by the ISCIII (PI2100989) and CIBERINFEC; the European Commission Horizon 2020 Framework Programme (grant numbers 731868 project VIRUSCAN FETPROACT-2016, and 101046084 project EPIC-CROWN-2); and the Fundación CaixaHealth Research (grant number HR18-00469 project StopEbola). Research in CNB-CSIC laboratory was funded by Fondo Supera COVID19 (Crue Universidades-Banco Santander) grant, CIBERINFEC, and Spanish Research Council (CSIC) grant 202120E079 (to J.G.-A.), CSIC grant 2020E84 (to M.E.), MCIN/AEI/10.13039/501100011033 (PID2020- 114481RB-I00 to J.G-A. and M.E.), and by the European CommissionNextGenerationEU, through CSIC’s Global Health Platform (PTI Salud Global) to J.G.-A. and M.E. Work in the CIB-CSIC laboratory was supported by MCIN/AEI/10.13039/501100011033 (PID2019-104544GB-I00 and 2023AEP105 to CA, and PID2020-113225GB-I00 to F.J.B.). Cryo-EM data were collected at the Maryland Center for Advanced Molecular Analyses which was supported by MPOWER (The University of Maryland Strategic Partnership). I.H.-M. receives the support of a fellowship from la Caixa Foundation (ID 100010434, fellowship code: LCF/BQ/IN17/11620074) and from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 71367. L.R.-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck.S

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2

17 p.-4 fig.Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT, are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT, the bispecific trimerbody TNTDNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNTDNGR-1, but not TNT, protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.SARS-CoV-2 B.1.351 and B.1.167.2 viruses used in this study were obtained through the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 653316. SARS-CoV-2 B.1 (MAD6 isolate) was kindly provided by José M. Honrubia and Luis Enjuanes (CNB-CSIC, Madrid, Spain). The authors thank Centro de Investigación en Sanidad Animal (CISA)-Instituto Nacional de Investigaciones Agrarias (INIA-CSIC) (Valdeolmos, Madrid, Spain) for the BSL-3 facilities. Research in LA-V laboratory was funded by the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 y COVID-19); the MCIN/AEI/10.13039/501100011033 (PID2020-117323RB-I00 and PDC2021-121711-I00), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (DTS20/00089), partially supported by the ERDF, the Spanish Association Against Cancer (AECC 19084); the CRIS Cancer Foundation (FCRIS-IFI-2018 and FCRIS-2021-0090), the Fundación Caixa-Health Research (HR21-00761 project IL7R_LungCan), and the Comunidad de Madrid (P2022/BMD-7225 NEXT_GEN_CART_MAD-CM). Work in the DS laboratory was funded by the CNIC; the European Union's Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator Grant 725091; MCIN/AEI/10.13039/501100011033 (PID2019-108157RB); Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); Atresmedia (Constantes y Vitales prize); Fondo Solidario Juntos (Banco Santander); and “La Caixa” Foundation (LCF/PR/HR20/00075). The CNIC was supported by the ISCIII, the MCIN and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S funded by MCIN/AEI/10.13039/501100011033). Research in RD laboratory was supported by the ISCIII (PI2100989) and CIBERINFEC; the European Commission Horizon 2020 Framework Programme (grant numbers 731868 project VIRUSCAN FETPROACT-2016, and 101046084 project EPIC-CROWN-2); and the Fundación Caixa-Health Research (grant number HR18-00469 project StopEbola). Research in CNB-CSIC laboratory was funded by Fondo Supera COVID-19 (Crue Universidades-Banco Santander) grant, CIBERINFEC, and Spanish Research Council (CSIC) grant 202120E079 (to J.G.-A.), CSIC grant 2020E84 (to M.E.), MCIN/AEI/10.13039/501100011033 (PID2020-114481RB-I00 to J.G-A. and M.E.), and by the European Commission-NextGenerationEU, through CSIC's Global Health Platform (PTI Salud Global) to J.G.-A. and M.E. Work in the CIB-CSIC laboratory was supported by MCIN/AEI/10.13039/501100011033 (PID2019-104544GB-I00 and 2023AEP105 to CA, and PID2020-113225GB-I00 to F.J.B.). Cryo-EM data were collected at the Maryland Center for Advanced Molecular Analyses which was supported by MPOWER (The University of Maryland Strategic Partnership). I.H.-M. receives the support of a fellowship from la Caixa Foundation (ID 100010434, fellowship code: LCF/BQ/IN17/11620074) and from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 71367. L.R.-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck.Peer reviewe

Distribution of primate and rodent OR orthologs with known ligands among OR classes and families.

<p>(A) Unrooted tree of human ORs based on similarity of amino acid properties. 12 human-chimpanzee-rhesus macaque orthologs, 5 human-chimp orthologs and 1 human-macaque ortholog are highlighted in orange. (B) Unrooted tree of mouse ORs based on similarity of amino acid properties. 17 mouse-rat orthologs are highlighted in blue. Receptor similarity was quantified using Grantham's amino acid property scale <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821-Grantham1" target="_blank">[29]</a>.</p

Cell surface expression does not predict function of ORs.

<p>(A) Primate OR2W1 orthologs and 2W subfamily members to allyl phenyl acetate. (B) Quantification of live cell-surface expression of each receptor. ** p<0.01 when compared to hOR2W1. Y-axis denotes the average Cy3 intensity in arbitrary units (a.u.) (n = 3, ± S.E.M.). S6 is positive control and Rho-pCI is negative control. (C) Representative image of live cell-surface staining for each receptor. For additional live-cell surface staining, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821.s013" target="_blank">Figure S13</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821.s020" target="_blank">Table S7</a>.</p

Dose-response curves of OR orthologs and paralogs to a given ligand.

<p>(A) OR5K1 orthologs and 5 K subfamily to eugenol methyl ether. (B) OR8D1 orthologs and 8D subfamily members to 4,5-dimethyl-3-hydroxy-2,5-dihydrofuran-2-one. (C) OR2J2 orthologs and 2J subfamily members to 1-octanol. X-axis is the concentration of a given odor in Log Molar. Y-axis is normalized response (n = 3, ± S.E.M.). Human (h), chimpanzee (c) and rhesus macaque (m), mouse receptors (m+number); <i>para</i> indicates a receptor that is a paralog to the human reference OR. Vector control is Rho-pCI. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821.s011" target="_blank">Figure S11</a> for additional dose-response data from orthologs and subfamily members.</p

Sequence similarity does not accurately predict the functional properties of ORs.

<p>For each OR pair, the (A) Jukes-Cantor (nucleotide) distance (B) Grantham (amino acid) distance and (C) <i>ω</i> (dN/dS) is plotted against the functional distance, as defined by the correlation (1-R, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821.s015" target="_blank">Table S2</a>) of the response across the 42-odor panel for ORs responding to more than three odors. <i>ω</i> values for human-chimp OR2W1 were included in the calculation but eliminated from the plot for better visual representation. Values closer to zero are more similar in sequence and function. Jukes-Cantor and <i>ω</i> do not correlate with functional distance (J-C r_s = 0.14, p = 0.36; <i>ω</i>, r_s = 0.18, p = 0.24) while Grantham's distance has a correlation to functional distance (r_s = 0.38, p = 0.01).</p

Comparison of amino acid similarity among orthologs and paralogs.

<p>Grantham's distance was calculated for OR open reading frame (ORF) and for 22 predicted binding residues (22AA) used in Man et al. (2004) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821-Man2" target="_blank">[17]</a>. (A) Orthologs are paralogs are significantly different using both ORF and 22AA (z = −6.61, p<0.0001 ORF; z = −7.35, p<0.0001 22AA, Wilcoxon Rank Sum). (B) Grantham's distance ORF and 22AA for orthologs and paralogs segregated by response to a common odor. Amino acid similarity of the 22 predicted binding residues (22AA) was significantly different for paralogs, with responding paralogs being more similar in sequence (z = −3.54, p<0.0004, Wilcoxon Rank Sum). n.s. is not significant. Box plots show minimum values,10% and 25% quantiles, median, 75% and 90% quantiles, and maximum values for each data set. Each ortholog and paralog is compared to the reference human OR in that group (listed first in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821.s019" target="_blank">Table S6</a>).</p

Response of OR orthologs to 42 chemically diverse odors.

<p>(A) Tuning curves of human, chimp and macaque OR2W1 orthologs tested against 42 odors using a cAMP-mediated luciferase assay <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821-Zhuang3" target="_blank">[26]</a>. Odorants are ordered along the x-axis according to the response elicited from the human OR2W1, with the best ligands closer to the center. Y-axis represents the luciferase response to an odor at 100 µM (n = 3, ± S.E.). Negative values on the y-axis indicate the odor elicited an inhibitory response on OR signaling. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821.s006" target="_blank">Figure S6</a> for additional tuning curves. (B) Response of chimp and macaque OR2W1 orthologs (variant responses) plotted against the human OR2W1 response using the data from (A). X-axis and y-axis are response in luciferase assay at 100 µM (n = 3, ± S.E.). The black line represents the unit-slope line. Odor abbreviations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821.s016" target="_blank">Table S3</a>.</p

Dose-response curves of an OR ortholog set to a given ligand.

<p>(A) Primate OR2W1 orthologs to allyl phenyl acetate. (B) Primate OR10G7 orthologs to eugenol. (C) Primate OR8K3 orthologs to (+)-menthol. (D) Rodent 268-1 orthologs to 1-octanol. <i>(E)</i> Rodent 272-1 orthologs to (+)-carvone. X-axis is the concentration of a given odor in Log Molar. Y-axis is normalized response (n = 3, ± S.E.M.). Human (h), chimpanzee (c) and rhesus macaque (m) in primate ortholog sets; mouse (m) and rat (r) for rodent ortholog sets. Vector control is Rho-pCI. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821.s009" target="_blank">Figure S9</a> for additional dose-response data.</p

Classification of functional changes in dose-response between orthologous OR pairs.

<p>(A) human-chimp, (B) human-macaque, (C) chimp-macaque and (D) mouse-rat. Using both the potency (EC50) and efficacy (dynamic range) to a particular ligand, an orthologous pair is classified as either indistinguishable, hyper/hypo functional (one OR had both a lower potency and efficacy) or undefined (orthologs were significantly different but potency and efficacy did not change concordantly). Each pie chart refers to the first species in the comparison (<i>e.g.</i> (A) human is ___ to chimp). For visual explanation, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002821#pgen.1002821.s008" target="_blank">Figure S8</a>.</p