key: cord-0951130-5nindbx8
authors: Ullah, Irfan; Prévost, Jérémie; Ladinsky, Mark S.; Stone, Helen; Lu, Maolin; Anand, Sai Priya; Beaudoin-Bussières, Guillaume; Symmes, Kelly; Benlarbi, Mehdi; Ding, Shilei; Gasser, Romain; Fink, Corby; Chen, Yaozong; Tauzin, Alexandra; Goyette, Guillaume; Bourassa, Catherine; Medjahed, Halima; Mack, Matthias; Chung, Kunho; Wilen, Craig B.; Dekaban, Gregory A.; Dikeakos, Jimmy D.; Bruce, Emily A.; Kaufmann, Daniel E.; Stamatatos, Leonidas; McGuire, Andrew T.; Richard, Jonathan; Pazgier, Marzena; Bjorkman, Pamela J.; Mothes, Walther; Finzi, Andrés; Kumar, Priti; Uchil, Pradeep D.
title: Live Imaging of SARS-CoV-2 Infection in Mice Reveals that Neutralizing Antibodies Require Fc Function for Optimal Efficacy
date: 2021-08-18
journal: Immunity
DOI: 10.1016/j.immuni.2021.08.015
sha: f44f1833838c1abbb862324b65db8b3d43abd383
doc_id: 951130
cord_uid: 5nindbx8

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

neutralized SARS-CoV-2 MA10 with similar efficacies to the WA1 strain in vitro (IC 50 = 0.01837 291 µg/mL). Though we had to use 5-fold more virus and older B6 mice (12-14 weeks) to induce 292 mortality, we observed a similar Fc effector requirement for CV3-1 to prophylactically protect 293 mice and reduce viral loads and inflammation in lung and brain ( Figure S6J-O) . The 294 requirement for Fc-effector function for CV3-1 prophylaxis suggested that the same should be 295 critical for CV3-1 therapy ( Figure 6A) . Indeed, while CV3-1 treatment at 3 dpi controlled 296 J o u r n a l P r e -p r o o f infection, cohorts treated with CV3-1 LALA displayed rapidly spreading lung infection and fully 297 succumbed by 6 dpi after an accelerated loss in body weight ( Figure 6B-F) . High viral loads 298 and enhanced cytokine mRNA expression in tissues also reflected the failure of the LALA NAbs 299 to treat pre-established viral infection ( Figure 6G) . Notably, while the lung viral loads in CV3-1 300 LALA cohorts were similar to that in the control, inflammatory cytokine mRNA in lungs, CXCL10 301 in particular, was significantly higher (Figure 6H-I) . Thus, in addition to Fab-mediated 302 neutralizing activity, Fc-FcR interactions were crucial for efficient clearing of infected cells and 303 curbing a cytokine-storm like phenotype. 304 305

Fc can recruit NK cells, monocytes, or neutrophils to facilitate clearance of infected cells and 308 shape the cytokine response produced by these cells for enhancing adaptive and cell-mediated 309 immune responses (Lu et al., 2018) . When NK cells were depleted prior to CV3-1 prophylaxis 310 ( Figure S7J, K) , weak nLuc signals appeared in lungs of infected mice; however, this did not 311 equal the flux intensities in control cohorts with no CV3-1 treatment ( Figure S7A-D) . They also 312 experienced a temporary but significant decrease in body weight ( Figure S7E ). Nevertheless, 313 NK cell depletion did not decrease the survival statistics of CV3-1 prophylaxis and all the mice 314 survived despite marginal increases in viral loads in target organs and significant increases in 315 pro-inflammatory cytokine mRNA expression ( Figure S7F-K) . Thus, while NK cells do 316 contribute to in vivo efficacy of CV3-1, their absence did not compromise the protection offered 317 by CV3-1 prophylaxis. 318

In therapeutic regimen format, when CV3-1 treatment was initiated at 3 dpi ( Figure 7A) , 319 depleting NK cells compromised protective efficacy with 25% of the mice succumbing to SARS-320

CoV-2 infection compared to a 100% survival rate of mice cohorts replete with NK cells ( Figure  321 7B-F, S7J, K). Depleting Ly6G + neutrophils and Ly6C hi CD11b + classical monocytes under CV3-322 1 therapy. resulted in 75% and 80% of the mouse cohorts respectively failing to control SARS-323

CoV-2 spread, with loss in body weight resulting in death ( Figure 7A -G, S7L-O) (Mack et al., 324 2001 ). This was accompanied by increased viral burden and enhanced expression of CCL2, 325 CXCL10, and Il6 mRNA in target tissues ( Figure 7H-J) . Overall, neutrophils, monocytes and NK 326 cells contributed to the antibody-dependent cure of mice from lethal SARS-CoV-2 infection and 327 were critical for the success of SARS-CoV-2 NAb-directed therapies. 328 329 Discussion 330

NAb therapies are being explored to augment current vaccination strategies against 331 SARS-CoV-2 to expand the protection afforded towards emerging variants of concern. 332 However, prior evidence for antibody-dependent enhancement of pathology caused by 333 respiratory viruses like RSV and SARS-CoV-1 warrants careful investigation of antibody effects 334 in vivo before clinical implementation (Iwasaki and Yang, 2020; Klasse and Moore, 2020). We 335 have established a whole-body imaging approach to follow the dynamics and pathogenesis of 336 SARS-CoV-2 infection in mice to facilitate preclinical studies for identifying effective therapeutic 337 measures against COVID-19. Temporal tracking revealed that SARS-CoV-2 first replicates in 338 the nasal cavity, reaches the lungs at 1 dpi where the infection expands till 3 dpi before 339 spreading systemically to other organs including the brain at 4 dpi. BLI also helped illuminate 340 how the highly potent human NAbs CV3-1 (targets Spike RBD) and CV3-25 (binds S2 domain) 341 differed in their ability to protect or treat SARS-CoV-2 infection in the highly susceptible K18-342 hACE2 mouse model. Imaging analyses revealed widespread distribution of NAb within the 343 animals, including in the nasal cavity and lungs where the virus infection is initially established, 344 and persistence for at least a week after administration, features that were critical for efficacy in 345 this acute model for SARS-CoV-2. BLI also revealed a therapeutic window of 3 dpi for CV3-1 346 NAb to successfully halt progression of infection from lungs to distal tissues. As previously 347 reported SARS-CoV-2 NAbs have a therapeutic window of 1 dpi (Alsoussi et Our data also establishes that neutralizing capacity of NAbs alone is insufficient to 361 garner clinical protection. LALA variants of CV3-1 revealed a crucial role for Fc-mediated 362 interactions in augmenting in vivo protection not only for therapy, but also in prophylaxis in 363 contrast to a recent report where Fc-effector was involved only during NAb therapy (Winkler et Scale bars in (B) and (F) denote radiance (photons/sec/cm 2 /steradian). p values obtained by 456 non-parametric Mann-Whitney test for pairwise comparison. * , p < 0.05; * * , p < 0.01; * * * , p < 457 0.001; * * * * , p < 0.0001; ns, not significant; Mean values ± SD are depicted. 458

See also Table S1, Figure S1 and Video S1 459 (12.5 mg/kg body weight) or as a 1:1 cocktail (6.25 mg/kg body weight each) 1 day prior to 558 challenging K18-hACE2 mice (i.n.) with SARS-CoV-2-nLuc followed by non-invasive BLI every 2 559 days. Human IgG1-treated (12.5 mg/kg body weight) mice were the control cohort (Iso). Requests for resources and reagents should be directed to and will be fulfilled by the Lead 665

Contact, Pradeep Uchil (pradeep.uchil@yale.edu). 666

All unique reagents generated in this study are available from the lead contact with a completed 668

Materials Transfer Agreement. 669 Indicated organs (nasal cavity, brain, lungs from infected or uninfected mice were collected, 858 weighed, and homogenized in 1 mL of serum free RPMI media containing penicillin-859 streptomycin and homogenized in 2 mL tube containing 1.5 mm Zirconium beads with BeadBug 860 6 homogenizer (Benchmark Scientific, TEquipment Inc). Virus titers were measured using three 861 highly correlative methods. Frist, the total RNA was extracted from homogenized tissues using 862

RNeasy plus Mini kit (Qiagen Cat # 74136), reverse transcribed with iScript advanced cDNA kit 863 (Bio-Rad Cat #1725036) followed by a SYBR Green Real-time PCR assay for determining 864 copies of SARS-CoV-2 N gene RNA using primers SARS-CoV-2 N F: 5'-865 ATGCTGCAATCGTGCTACAA-3' and SARS-CoV-2 N R: 5'-GACTGCCGCCTCTGCTC-3'. 866

Second, serially diluted clarified tissue homogenates were used to infect Vero-E6 cell culture 867 monolayer. The titers per gram of tissue were quantified using standard plaque forming assay 868 described above. Third, we used nanoluciferase activity as a shorter surrogate for plaque assay. Assay mouse primers FAM-GAPDH, HEX-IL6, TEX615-CCL2, Cy5-CXCL10,  884 and Cy5.5-IFNgamma. The reaction plate was analyzed using CFX96 touch real time PCR 885 detection system. Scan mode was set to all channels. The PCR conditions were 95 °C 2 min, 40 886 cycles of 95 °C for 10 s and 60 °C for 45 s, followed by a melting curve analysis to ensure that 887 each primer pair resulted in amplification of a single PCR product. mRNA copy numbers of Il6, 888

Ccl2, Cxcl10 and Ifng in the cDNA samples of infected mice were normalized to Gapdh mRNA 889 with the formula ΔC t (target gene)=C t (target gene)-C t (Gapdh). The fold increase was determined 890 using 2 -ΔΔCt method comparing treated mice to uninfected controls. 891 892

For evaluating the effect of NK cell depletion during CV3-1 prophylaxis, anti-NK1.1 (clone 894 PK136; 12.5 mg/kg body weight) or an isotype control mAb (BioXCell; clone C1.18.4; 12.5 895 mg/kg body weight) was administered to mice by i.p. injections every 2 days starting at 48 h 896 before SARS-CoV-2-nLuc challenge till 8 dpi. The mice were bled after two days of antibody 897 depletion, necropsy or at 10 dpi (surviving mice) for analyses. To evaluate the effect of NK 898 cell and neutrophil depletion during CV3-1 therapy, anti-NK1.1 (clone PK136; 12.5 mg/kg body 899 weight) or anti-Ly6G (clone: 1A8; 12.5 mg/kg body weight) was administered to mice by i.p 900 injection every two days starting at 1 dpi respectively. Rat IgG2a mAb (BioXCell; clone C1.18.4; 901 12.5 mg/kg body weight) was used as isotype control. The mice were sacrificed and bled at 10 902 dpi for analyses. For evaluating the effect of monocyte depletion on CV3-1 therapy, anti-CCR2 903 (clone MC-21; 2.5 mg/kg body weight) (Mack et al., 2001) or an isotype control mAb (BioXCell; 904 clone LTF-2; 2.5 mg/kg body weight) was administered to mice by i.p injection every two days 905 starting at 1 dpi. The mice were sacrificed and bled 2-3 days after antibody administration or at 906 10 dpi to ascertain depletion of desired population. identified as CD3-NK1.1+ cells using PE/Cy7 anti-mouse CD3(17A2) and APC anti-mouse NK-915 1.1 (PK136). Neutrophils were identified as CD45 + CD11b + Ly6G + cells using APC Rat anti-916 mouse CD45 (30-F11), PE anti-mouse CD11b (M1/70) APC/Cy7 and anti-mouse Ly-6G (1A8). 917

Ly6C hi monocytes were identified as CD45 + CD11b + Ly6C hi cells using APC Rat anti-mouse 918 CD45 (30-F11), PE anti-mouse CD11b (M1/70) and APC/Cy7 anti-mouse Ly-6C (HK1.4). Data 919 were acquired on an Accuri C6 (BD Biosciences) and were analyzed with Accuri C6 software. 920

FlowJo software (Treestar) was used to generate FACS plot shown in Figure S7 . 100,000 -921 200,000 viable cells were acquired for each sample. 922 923

Lung, brain and testis tissue samples from hACE2 transgenic mice challenged intranasally with 925 SARS-CoV-2-nLuc (1 x 10 5 FFU; 6 dpi) were imaged after necropsy using bioluminescence 926 imaging (IVIS, Perkin Elmer), pruned to isolate regions with high nLuc activity and immediately 927 pre-fixed with 3% glutaraldehyde, 1% paraformaldehyde, 5% sucrose in 0. Thermo-Fisher, Cat. #16140-071), 1% HEPES Buffer Solution (15630-130), and 1% penicillin-936 streptomycin (Thermo Fisher, Cat. #15140-122). Cells were grown in a humidified incubator at 937 37 ºC with 5 % CO 2 . Vero E6 cells were seeded into six well dishes and infected with SARS-938

CoV-2 at a multiplicity of infection of 0.01 for 48 hours before fixing and preparing for electron 939 microscopy. Cells were pre-fixed with 3% glutaraldehyde, 1% paraformaldehyde, 5 % sucrose in 940 0.1M sodium cacodylate trihydrate, removed from the plates and further prepared by high-941 pressure freezing and freeze-substitution as described below. 942

Tissues samples were further cut to ~0.5 mm 3 blocks and cultured cells were gently pelleted. 943

Both samples were rinsed with fresh cacodylate buffer and placed into brass planchettes (Type 944 A; Ted Pella, Inc., Redding, CA) prefilled with 10% Ficoll in cacodylate buffer. The tissues were 945 covered with the flat side of a Type-B brass planchette and rapidly frozen with an HPM-010 946 high-pressure freezing machine (Leica Microsystems, Vienna Austria). The frozen samples 947

were transferred under liquid nitrogen to cryotubes (Nunc) containing a frozen solution of 2.5% 948 osmium tetroxide, 0.05 % uranyl acetate in acetone. Tubes were loaded into an AFS-2 freeze-949 substitution machine (Leica Microsystems) and processed at -90°C for 72 h, warmed over 12 h 950 to -20°C, held at that temperature for 6 h, then warmed to 4°C for 2 h. The fixative was 951 removed, and the samples rinsed 4 x with cold acetone, following which they were infiltrated 952 with Epon-Araldite resin (Electron Microscopy Sciences, Port Washington PA) over 48 h. The 953 spleen tissue was flat-embedded between two Teflon-coated glass microscope slides. Resin 954 was polymerized at 60°C for 48 h. 

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al., 2020; Turonova et al., 2020; Yao et al., 2020) . These were compared to identified virions 984 within SARS-CoV-2-infected cultured Vero E6 cells that had been prepared for EM by the same 985 methodology ( Figure 2O -Q). We used the following criteria to positively identify SARS-CoV-2 986 virions in tissues: (i) Structures that were spherical in 3D with ~60-120 nM diameters and were 987 not continuous with other adjacent structures, (ii) Spherical structures with densities 988 corresponding to a distinct membrane bilayer, internal puncta consistent with ribonucleoproteins 989 (Yao et al., 2020) , and densities corresponding to surface spikes on the external peripheries of 990 the spheres. In further characterization of virions, we noted that the inner vesicles of 991 multivesicular bodies (MVBs) have been mis-identified as SARS-CoV-2 by electron microscopy 992 (Calomeni et al., 2020) . We therefore compared measurements of MVB inner vesicles and carbon-coated, glow-discharged 100-mesh copper/rhodium grids (Electron Microscopy 1009 Sciences). Grids were incubated 1 hr with 10% calf serum in PBS to block nonspecific antibody 1010 binding, then incubated 2 hrs with anti-S antiserum (Cohen et al., 2021) . Mosaic nanoparticles 1011 elicit cross-reactive immune responses to zoonotic coronaviruses in mice (Cohen et al., 2021) . 1012 diluted 1:500 in PBS with 5% calf serum. Grids were rinsed (4x 10') with PBS then labeled for 2 1013 hrs with 10 nm gold conjugated goat anti-mouse secondary antibody (Ted Pella, Inc.). Grids 1014 were again rinsed (4x 10') with PBS, then 3x with distilled water and negatively stained with 1% 1015 uranyl acetate in 1% methylcellulose (Sigma) for 20'. Grids were air-dried in wire loops and 1016 imaged as described for ET. 1017

Site-directed mutagenesis was performed on plasmids expressing CV3-1 and CV3-25 antibody 1020 heavy chains in order to introduce the LALA mutations (L234A/L235A) or the GASDALIE 1021 mutations (G236A/S239D/A330L/I332E) using to the QuickChange II XL site-directed 1022 mutagenesis protocol (Stratagene). The plates were subsequently centrifuged for 1 min at 300xg, and incubated at 37°C, 5% CO2 1202 for 5 hours before being fixed in a 2% PBS-formaldehyde solution.