key: cord-0801035-9il8zayx authors: Winkler, Emma S.; Gilchuk, Pavlo; Yu, Jinsheng; Bailey, Adam L.; Chen, Rita E.; Zost, Seth J.; Jang, Hyesun; Huang, Ying; Allen, James D.; Case, James Brett; Sutton, Rachel E.; Carnahan, Robert H.; Darling, Tamarand L.; Boon, Adrianus C. M.; Mack, Matthias; Head, Richard D.; Ross, Ted M.; Crowe, James E.; Diamond, Michael S. title: Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions and monocytes for optimal therapeutic protection date: 2020-12-28 journal: bioRxiv DOI: 10.1101/2020.12.28.424554 sha: 49280157122513b6098b78d6e9aeb6766993589d doc_id: 801035 cord_uid: 9il8zayx SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes for therapeutic efficacy. Thus, potently neutralizing mAbs require Fc effector functions for maximal therapeutic benefit during therapy to modulate protective immune responses and mitigate lung disease. Effects on SARS-CoV-2-induced weight loss were comparable between COV2-2050 or 123 COV2-2050 LALA-PG at all doses with a loss of protection observed only at the 1.6 μg (0.08 124 mg/kg) dose (Fig 1E-F) . Levels of COV2-2050 correlated with clinical protection (Fig 1G-H (Fig 2A) . The 145 differences in protection were not due to disparate levels of intact COV2-2050 and COV2-2050 146 8 LALA-PG variants, as equivalent amounts were detected in serum at 0 and 4 dpi (Fig 2B) . Treatment at 1 dpi with COV2-2050, but not COV2-2050 LALA-PG, reduced SARS-CoV-2 viral 148 RNA levels in the lung at 4 and 8 dpi substantially as measured by qRT-PCR (Fig 2C-D) . In 149 contrast, D+2 treatment with either intact COV2-2050 or COV2-2050 LALA-PG did not reduce 150 viral RNA levels in the lung when compared to isotype-treated controls. However, COV2-2050 151 and COV2-2050 LALA-PG mAbs both reduced levels of infectious virus in the lung at 4 dpi (Fig 152 2E ). Given the disparity in viral RNA levels, this effect might be due in part to ex vivo 153 neutralization after lung tissue homogenization, as reported for other respiratory viruses 154 (Subbarao et al., 2004; Wells et al., 1981) . Although D+2 treatment did not reduce viral RNA 155 levels in the lung, K18-hACE2 mice receiving intact COV2-2050 at D+2, but not COV2-2050 156 LALA-PG, showed functional improvement in pulmonary mechanics including inspiratory 157 capacity, respiratory resistance, elastance, tissue damping, and compliance (Fig 2F) . These 158 results correlated with a smaller downward deflection of the pressure-volume loop and improved 159 lung compliance and distensibility (Fig 2G) . To corroborate these results, we tested the therapeutic activity of additional neutralizing 161 human mAbs that bind the RBD on the S protein, COV2-3025, COV2-2072, and COV2-2381 162 (Zost et al., 2020b) , and compared them to corresponding LALA-PG or LALA variants of each 163 mAb. For COV2-2381, we used a LALA variant of COV2-2381, since this version also is 164 commonly used to minimize Fc effector functions (Hessell et al., 2007; Sapparapu et al., 2016) . The LALA form lacks the P329G mutation, which is needed to fully abolish binding to all mouse CoV-2 equivalently compared to their respective LALA-PG or LALA variants (Fig 2H, S1A , and 169 S1E) and introduction of the LALA mutation abolished binding to mouse FcγRI and FcγRIV (Fig 170 S1B ). Administration of intact COV2-3025 at D+1 recapitulated the same pattern of protection 9 observed with COV2-2050 treatment: COV2-3025 LALA-PG failed to protect mice from weight 172 loss or reduce viral titers compared to the intact COV2-3025 (Fig 2I-J) . In comparison, COV2-2381 LALA partially protected against weight loss, and viral 174 burden was reduced in mice receiving either COV2-2381 or COV2-2381 LALA compared to 175 isotype-treated controls, although we noted a trend towards lower levels of viral RNA in animals 176 receiving the intact COV2-2381 (Fig S1C-D) . Administration of COV2-2072 LALA-PG at D+1 177 partially protected against SARS-CoV-2-induced weight loss compared to the isotype-treated 178 controls, but failed to reduce viral titers, unlike the intact COV2-2072 (Fig S1F-G) . The relative However, at 8 dpi, we observed a significant reduction in the numbers of CD45 + cells, 211 neutrophils, and CD8 + T cells in mice treated at D+1 with COV2-2050 compared to COV2-2050 212 LALA-PG (Fig 3D) , consistent with histopathological findings (Fig 3A) . However, the only 213 difference in immune cell infiltrates in BAL fluid at D+2 was a reduced number of alveolar 214 macrophages in animals receiving COV2-2050 compared to COV2-2050 LALA-PG (Fig 3D) . Given that viral RNA levels were equivalent in mice receiving COV2-2050 and COV2-2050 216 LALA-PG at D+2 (Fig 2C and E) and only small differences in BAL cell number were observed, PG-treated mice were more similar to isotype control mAb-treated animals (Fig 4A) . Indeed, and Jak3), type I IFN signaling (e.g., Ifnar2, Stat2, Ifit1, Ifit2, Ifit3, and Irf7), and leukocyte 236 chemotaxis (e.g., Ccl2, Cxcl10, and Ccl7) as well as genes involved in cell proliferation (e.g., Egfr, Fgfr1, Fosl1, Myc, and Cdkn2b) and metalloproteinase-mediated extracellular matrix 238 organization (e.g., Adam15, Adam19, Col1a, Mmp14, and Itgam) (Fig S4 and Table S1 ). We next performed CompBio analysis (v2.0, PercayAI), as we did previously (Adamo et Table S2 ). Pathways unique to the COV2-2050 D+1 treatment group compared to the 243 isotype-treated group included genes involved in actinomyosin-associated cell adhesion (e.g., Kif1c, Ctnnd1, Nectin3, Unc45b, and Prkca) and Rho GTPase signaling (e.g., Rhoq, Mapk3, 245 Prkca, and Cdc42bpa), processes that are typically associated with wound repair programs 246 (Verboon and Parkhurst, 2015) (Fig 4D) . The expression pattern of these gene sets in the 247 COV2-2050 D+1 treatment was similar to naïve animals, suggesting that the intact antibody 12 limited virus-induced perturbations in transcription. Pathways uniquely downregulated in the 249 COV2-2050 D+1 group included genes involved in type I IFN and NFkB-dependent signaling 250 (e.g., Irf7, Stat2, Nfkb2, Bst2, Isg15, and Ikbk3), which may in part be due to the lower levels of 251 viral RNA detected (Fig 2C-D and 4D ). Pathways that were downregulated in the D+2 COV2-2050-treated group compared to 253 the isotype control or D+2 COV2-2050 LALA-PG-treated animals included S100A8-associated 254 innate immune signaling (e.g., Reg3g, Saa3, Itgma, Mmp8, and S100a8), oncostatin M receptor 255 associated signaling (e.g., Il6, Osmr, Csf1, and Socs3), and extracellular matrix remodeling 256 (e.g., Adamts15, Col5a1, Vcam1, and Lama4) (Fig S5, (Fig 5A and S6) , natural killer (NK) cells (anti-NK1.1) (Fig 5B and S6 ), or neutrophils (anti-Ly6G) (Fig 5C and S6 ) in combination with COV2-2050 treatment at D+1. Depletion of neutrophils or NK cells did not affect SARS-CoV-2 pathogenesis in the presence of 274 COV2-2050 or the isotype control mAb (Fig 5B and C) . However, when monocytes were 275 depleted, COV2-2050 failed to prevent the weight loss phenotype seen in non-depleted, COV2-276 2050-treated mice (Fig 5A) . Moreover, monocyte depletion in the setting of COV2-2050 therapy 277 was associated with greater immune cell infiltration and lung damage (Fig 5D) . Indeed, 278 expression of Ccl2, Cxcl10, and Il6 were increased following COV2-2050 treatment and 279 monocyte depletion compared to treatment with COV2-2050 or an isotype control mAb (Fig 5E) . The monocyte depletion phenotype, however, was not associated with changes in viral RNA 281 levels at 8 dpi (Fig 5F) . This result suggests that monocytes are a key immune cell type that 282 mediates therapeutic antibody-dependent protection through a mechanism that at least is 283 partially independent of viral clearance. Consistent with observations in mice, passive transfer of intact COV2-2050 prevented weight 294 loss compared to isotype mAb-treated animals at 5 and 6 dpi, and this protection was lost in 295 animals treated with the COV2-2050 LALA-PG variant (Fig 6A) . Furthermore, hamsters treated 296 with intact COV2-2050, but not COV2-2050 LALA-PG, showed a reduction in viral RNA levels at 297 6 dpi (Fig 6B) . The improved viral burden with COV2-2050 was associated with lower levels of 298 the inflammatory mediators Cxcl10, Ccl2, Ccl3, Ccl5, and Ifit3 (Fig 6C) . Thus, therapeutic For BAL staining, cells were gated to live, single, autofluorescent-negative CD45 + cells to 632 identify hematopoietic cells. Alveolar macrophages were identified as SiglecF hi CD11c hi cells. Neutrophils were identified as Ly6G hi CD11b hi cells. CD11bcells were gated further into CD4 + 634 and CD8 + T cells. CD11b hi Ly6Gcells were gated subsequently using CD64, CD24, and MHC-II. MHCII hi CD24 hi were defined as CD11b + DCs. MHCII lo Ly6 hi cells were defined as monocytes. Representative flow cytometry plots of monocytes and neutrophils from peripheral blood at 8 dpi 658 following intraperitoneal injection of a depleting anti-CCR2 mAb or isotype control mAb. B. Frequency of Ly6C hi monocytes and neutrophils in blood at 8 dpi following anti-CCR2 or isotype ANOVA. Changes in functional parameters or immune parameters were compared to isotype-907 treated animals and were analyzed by one-way ANOVA or one-way ANOVA with Dunnett's test. 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