key: cord-0934584-ufj7r0qy
authors: Wang, Shuang; Peng, Yun; Wang, Rongjuan; Jiao, Shasha; Wang, Min; Huang, Weijin; Shan, Chao; Jiang, Wen; Li, Zepeng; Gu, Chunying; Chen, Ben; Hu, Xue; Yao, Yanfeng; Min, Juan; Zhang, Huajun; Chen, Ying; Gao, Ge; Tang, Peipei; Li, Gang; Wang, An; Wang, Lan; Chen, Shuo; Gui, Xun; Zhang, Jinchao; Yuan, Zhiming; Liu, Datao
title: An antibody-dependent enhancement (ADE) activity eliminated neutralizing antibody with potent prophylactic and therapeutic efficacy against SARS-CoV-2 in rhesus monkeys
date: 2020-07-27
journal: bioRxiv
DOI: 10.1101/2020.07.26.222257
sha: 3364b2ae8c3f5302e4d2a8149cd558a4f7565993
doc_id: 934584
cord_uid: ufj7r0qy

Efficacious interventions are urgently needed for the treatment of COVID-19. Here, we report a monoclonal antibody (mAb), MW05, showing high SARS-CoV-2 neutralizing activity by disrupting the interaction of receptor binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2) receptor. Crosslinking of Fc with FcγRIIB mediates antibody-dependent enhancement (ADE) activity by MW05. This activity was eliminated by introducing the LALA mutation to the Fc region (MW05/LALA). Most importantly, potent prophylactic and therapeutic effects against SARS-CoV-2 were observed in rhesus monkeys. A single dose of MW05/LALA completely blocked the infection of SARS-CoV-2 in a study of its prophylactic effect and totally cleared SARS-CoV-2 in three days in a treatment setting. These results pave the way for the development of MW05/LALA as an effective strategy for combating COVID-19.

Accordingly, pre-incubation of MW05 with FcγRIA recombinant protein showed higher ADE 108 inhibition than did pre-incubation of irrelevant hIgG1with Raji cells by disruption of MW05 Fc 109 with with FcγRIIB on Raji cells (Fig.3 D and E) . To eliminate the risk of ADE and Fc-110 mediated acute lung injury in vivo, we introduced the LALA mutation to the Fc region of MW05 111 (MW05/LALA) to decrease the engagement of MW05 with FcγRs. This mutation completely 112 eliminated ADE activity of MW05 without decreasing its neutralizing activity (Fig. 3 F and G) . 113 We evaluated the prophylactic and therapeutic effects of MW05/LALA in a rhesus monkey 114 SARS-CoV-2 infection model. In the prophylactic (pre-challenge) group, three animals were 115 injected intravenously with a single dose of MW05/LALA (20 mg/kg) one day before receiving 116 a 1×10 5 50% tissue culture infectious dose (TCID50) SARS-CoV-2 challenge via intratracheal 117 incubation (Fig. 4A ). MW05/LALA antibody effectively protected animals from SARS-CoV-2 118 infection; almost no virus was detected in the oropharyngeal swabs of the prophylactic group 119 (Fig. 4B) . 120

In the treatment (post-challenge) group, three animals were first challenged with 1×10 5 121 TCID50 SARS-CoV-2. Then, at day 1 post infection (dpi), a single dose of MW05/LALA (40 122 mg/kg) was administered intravenously to these animals (Fig. 4A) . Animals in the control group 123 (n=3) were given a single dose of irrelevant hIgG1 (20 mg/kg) on 1 dpi. In the control group, 124 the viral loads in oropharyngeal swabs increased to a peak of about 10 7.0 RNA copies/mL on 4 125 dpi, then decreased to the limit of detection on 7 dpi (Fig. 4B) . Virus was only detected in the 126 rectal swabs of two animals in the control group (Fig. 4C) . Notably, virus titers decreased in 127 the MW05/LALA treatment group immediately after administration. No virus was detected in 128 the MW05/LALA treatment group even on 4 dpi, the time point at which viral titers in the 129 control group reached their peak. A single dose of MW05/LALA exhibited SARS-CoV-2 130 therapeutic efficacy in a rhesus monkey model, clearing virus in three days after antibody 131 administration (Fig. 4B) . No significant weight loss or body temperature change was observed 132 in any of the animals during the study (Extended Data Fig. 3 and 4) . No virus was detected in 133 nasal swabs or blood samples (Extended Data Fig. 5) . Additionally, no significant abnormal 134 hematology changes were observed (Extended Data Fig. 6) . 135

Rhesus monkeys challenged with SARS-CoV-2 were evaluated for tissue damage. One 136 monkey from each group was euthanized for necropsy on 6 and 7 dpi. Interstitial pneumonia 137 symptoms were observed in the control group, including thickened alveolar septa, intensive 138 infiltration of monocytes and lymphocytes, and proliferation of fibroblasts (Fig. 4D ). We also 139 observed cellulose exudation in some alveolar cavities, with the formation of hyaline membrane 140 and pulmonary hemorrhaging (Fig. 4D ). Monkeys in treatment group displayed limited 141 pathological lung changes, with overall alveolar structure intact and much lower levels of 142 fibroblasts proliferation and leukocyte infiltration than were observed in the control monkeys 143 Immunohistochemical analysis of virus in lung tissues showed that SARS-CoV-2 protein 148 only been detected in the lung tissue of the control group on 5 dpi but not 6 dpi and 7 dpi. In 149 comparison, viral proteins were undetectable in the lung tissues of animals in the prophylactic 150 and therapeutic groups (Fig. 4E) . In order to further understand the distribution of SARS-CoV-151 2 in upper respiratory tract, trachea and bronchus tissue samples were collected on 6 dpi and 7 152 dpi. Viral titers were then determined by qRT-PCR. On 6 dpi and 7 dpi, high levels of CoV-2 RNA copies were detected in trachea and bronchi tissues of control animals, while no 154 viral nucleic acid was detected from tissue samples of both prophylactic and therapeutic groups 155 (Fig. 4F) . 2 RBD mutants, SARS-CoV-2 S1, SARS-CoV S1 (Cat: 40150-V08B1, Sino Biological), MERS-204

CoV S1 (Cat: 40069-V08B1, Sino Biological)), the proteins were first coated on 96 well ELISA 205 plates at 1 µg/ml in 100 µL at 4℃ overnight. After blocking with 5% BSA in PBS, serially diluted 206 mAbs were added to the plates and incubated for 60 min at 37℃. Plates were washed and secondary 207

Ab Goat Anti-Human IgG Fc-HRP (Cat: 109-035-098, Jackson ImmunoResearch) was added. TMB 208 was used for color development and absorbance at 450 nm was measured using a microplate reader. 

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