key: cord-0715683-16g0y7tm
authors: Lee, Gunsup; Budhathoki, Shailesh; Choi, Hyeok Soon; Oh, Kwang-ji; Lee, Geum-Young; Ham, Yeon Kyoung; Kim, Young Jun; Lim, Ye Rin; Hoang, Phuong Thi; Lee, Yongjun; Lim, Seok-Won; Kim, Jun-Mo; Cho, Seungchan; Song, Jin-Won; Lee, Sukchan; Kim, Won-Keun
title: 3D8, a nucleic acid-hydrolyzing scFv, confers antiviral activity against SARS-CoV-2 and multiple coronaviruses in vitro
date: 2020-11-25
journal: bioRxiv
DOI: 10.1101/2020.11.25.398909
sha: 216805a13120cdc3fbc29c83d2e34e18e847cb55
doc_id: 715683
cord_uid: 16g0y7tm

The current pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose a critical public health threat worldwide. Coronaviruses (subfamily Orthocoronavirinae, family Coronaviridae, order Nidovirales) are a group of enveloped positive-sense single-stranded RNA viruses. Six pathogenic human coronaviruses, likely zoonotic viruses, cause the common cold in humans. A new emerging coronavirus, SARS-CoV-2, become a crucial etiology for the Coronavirus-induced disease 19 (COVID-19). However, effective therapeutics and vaccines against multiple coronaviruses remain unavailable. This study aimed to investigate an antiviral molecule, single chain variable fragment (scFv), against SARS-CoV-2 and other coronaviruses. 3D8, a recombinant scFv, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. Here, we report that 3D8 scFv inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the absence of coronavirus nucleoproteins and infectious particles in 3D8 scFv-treated cells, respectively. In addition, we observed the antiviral effects of 3D8 against HCoV-OC43 and PEDV. In conclusion, this study provides insights into the broad-spectrum antiviral agent of 3D8 scFv; thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses. Key points (Main message) 3D8, a nucleic acid-hydrolyzing scFv, exhibits potent prophylactic and therapeutic antiviral effects on SARS-CoV-2. 3D8 exhibits broad-spectrum antiviral activity against multiple coronaviruses: hCoV OC43 and PEDV. 3D8 potentially degrades viral RNA.

, which was identified as the novel coronavirus disease (COVID-19) caused 64 by SARS-CoV-2 (8). SARS-CoV-2 has several similarities to SARS-CoV and binds to the 65 common host cell receptor, angiotensin-converting enzyme 2 (ACE-2), and Transmembrane 66 Serine Protease 2 (TMPRSS2) (9, 10); however, the novel strain is genetically distinct from SARS-67 CoV-1 (11). The novel coronavirus SARS-CoV-2 is transmitted through species barriers from bats 68 to humans (12). COVID-19 is characterized by influenza-like symptoms ranging from mild to 69 4 severe lung injury as well as multi-organ failure, leading to death in patients with comorbidities 70 (13). This novel virus has led to a global pandemic, which resulted in unparalleled public health 71 emergencies (14). As of November 24, 2020, it has rapidly spread to 220 countries and territories, 72 infecting over 58.7 million people including more than 1.38 million deaths (15) 73 [https://covid19.who.int].

The rapid and widespread emergence of SARS-CoV-2 presents the urgent need for antiviral 75 countermeasures (4). Currently, there are no available therapeutics against human coronaviruses. Here, this study aimed to investigate the antiviral activity of 3D8 scFv against emerging 94 coronaviruses in vitro. These data provide insight into a broad-spectrum antiviral agent of scFv 95 against SARS-CoV-2 and multiplex coronaviruses. 

To determine the antiviral activity of the 3D8 scFv against SARS-CoV-2, different concentrations 100 of the scFv were applied to Vero E6 cells after virus challenge. SARS-CoV-2 replication in cultures 101 treated with various doses of 3D8 was quantified using RT-qPCR (Fig 1A) . The replication of 102 SARS-CoV-2 significantly decreased in a 3D8 dose-dependent manner. The 10 µM and 5 µM 103 concentrations of 3D8 effectively inhibited viral replication by up to approximately 90% and 75%, 104 respectively, compared to the non-treatment group. The production of infectious virus particles was 105 quantified by performing the plaque assay ( Fig 1B) . The viral titer of SARS-CoV-2 was reduced in 106 a 3D8 dose-dependent manner. In particular, when treated with 10 µM of 3D8, the titer of the virus 107 was reduced by 10 times compared to the non-treatment group. Continual treatment with 3D8 108 showed antiviral activity against SARS-CoV-2 at an effective concentration (EC50) of 4.25 µM 109 ( Fig 1C) . Moreover, this scFv did not show cytotoxicity in Vero E6 cells treated with the 3D8 scFv 110 at concentrations ranging from 1 µM to 10 µM ( Fig 1D) . However, cytotoxicity was noted at a dose We determined the prophylactic antiviral activity of the 3D8 scFv against SARS-CoV-2 in 115 pretreated cell cultures. A significant reduction in the SARS-CoV-2 N gene copies was observed 116 upon treatment with 10 µM 3D8 scFv (Fig 2A) . The gene copy number of the SARS-CoV-2 E gene 117 and RdRp gene was reduced by 99.6% and 99.4%, respectively (data not shown). The N protein of 118 SARS-CoV-2 was not observed after treatment with the 3D8 scFv ( Fig 2B) . Furthermore, we 119 determined the inhibitory effect of 3D8 on the production of infectious particles of SARS-CoV-2.

The production of infectious virus particles was more than 10 times lower in the scFv-treated group 121 than in the control group ( Fig 2C) . Collectively, these data demonstrated that 3D8 has a 122 prophylactic effect on SARS-CoV-2 infection.

To determine the therapeutic effect of 3D8 at 2 h post-infection (p.i.), we assessed the inhibitory 126 activity of SARS-CoV-2 based on the reduction of the relative gene copy number (Fig 2D) . 3D8 OC43 RNA was significantly reduced in a 3D8 dose-dependent manner (Fig 3A) . The expression 139 of viral proteins was inhibited upon treatment with 3D8 ( Fig 3B) . 3D8 effectively inhibited the 140 replication of HCoV-OC43 with the EC50 value of 1.40 µM (Fig 3C) . Immunohistochemistry 141 analysis exhibited a reduction in HCoV-OC43 replication upon 3D8 treatment (Fig 3D) . 142 Treatment with 3D8 resulted in the effective inhibition of viral replication upon PEDV infection.

The load of PEDV RNA was significantly suppressed in a 3D8 dose-dependent manner (Fig 4A) . 144 The expression of viral proteins was reduced upon treatment with 3D8 ( Fig 4B) . The EC50 value of 145 3D8 against PEDV was 1.10 µM (Fig 4C) . Immunohistochemistry analysis revealed a reduction in 146 PEDV replication upon 3D8 treatment (Fig 4D) . These data demonstrated the broad-spectrum 147 activity of 3D8 against multiple zoonotic coronaviruses. of 3D8 revealed a caveolin-dependent manner without a carrier (30). The intranasal transfer of 3D8 184 scFv into a mouse described the presence of the protein in the epithelial barrier of lung tissues (31).

Taken together, the 3D8, a nucleic-acid hydrolyzing mini-antibody, may be a potential candidate 186 for antivirals due to the broad spectrum, the easy penetration to the cell, and the accessibility to the Vero E6 cells were plated at 1 × 10 6 cells per well in 6-well plates (Corning) and incubated at 37 227 °C with 5% CO2. A confluent monolayer of cells was washed with phosphate-buffered saline (PBS, 228 Lonza, USA, BioWhittaker ® ), infected with ten-fold serial dilutions of viral suspension prepared in 229 11 serum-free maintenance media (DMEM only), and incubated at 37 °C. Following infection for 1 h 230 with intermittent shaking at 15-min intervals, the viral inoculum was aspirated, and overlay media 231 (DMEM/F12 media) containing 4% bovine serum albumin (BSA), 2 mM glutamine, 2.5% sodium 232 bicarbonate (NaHCO3), 10 mM HEPES, 50 mg/mL DEAE dextran, 100 U/mL penicillin, 100 233 μg/mL streptomycin (Pen Strep), and 0.6% immunodiffusion-grade Oxoid agar was added. After 234 4-5 days of incubation at 37 °C with 5% CO2, fixation with 4% paraformaldehyde (Biosesang, 235 F1119Z21 YR) was performed. After overnight incubation, the overlay agar media was flicked 236 using a metal spatula, and the plates were stained with crystal violet (0.1% crystal violet in 20% 237 methanol) for 10 min. Plaques were enumerated, and viral titers were quantified.

In vitro antiviral activity 240 Cells were seeded at 1 × 10 6 cells per well and allowed to adhere for 24 h at 37 °C with 5% CO2 in Following this, PEDV and hCoV-OC43 were incubated with TRITC-conjugated anti-mouse Ab

(1:500) (Abcam, #ab6786), and 3D8 was incubated with Alexa 488-conjugated anti-rabbit Ab

(1:1000) (Abcam, #ab150077). The nuclei were stained with Hoechst (Thermo Fisher, #62249) 295 during the last 10 min of incubation at room temperature. Cells were mounted in mounting medium 296 (VECTASHIELD, #H-1200) and observed using a NIKON A1R (Eclipse A1Rsi and Eclipse Ti-E). 307 The authors declare that the research was conducted in the absence of any commercial or financial 308 relationships that could be construed as a potential conflict of interest. 

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