Broad-Spectrum Antiviral Activity of 3D8, a Nucleic Acid-Hydrolyzing Single-Chain Variable Fragment (scFv), Targeting SARS-CoV-2 and Multiple Coronaviruses In Vitro

Abstract

The virus behind the current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of novel coronavirus disease (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single-chain variable fragment (scFv), a recombinant antibody, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. The antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses was evaluated in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. The nucleic acid-hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. We found that 3D8 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 reduction of coronavirus nucleoproteins and infectious particles, respectively, in 3D8 scFv-treated cells. These data demonstrate the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.

Keywords: 3D8 scFv; COVID-19; SARS-CoV-2; coronaviruses; single-chain variable fragment.

Figure 1

RNA-hydrolyzing activity of 3D8 for in vitro transcripts of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). (A) Nuclease activity of 3D8 against SARS-CoV-2. In vitro transcripts of SARS-CoV-2 were prepared and treated with 3D8. (B) Nuclease activity of 3D8 against HCoV-OC43. In vitro transcripts of HCoV-OC43 were prepared and treated with 3D8. (C) Nuclease activity of 3D8 against PEDV. In vitro transcripts of PEDV were prepared and treated with 3D8. Hydrolysis activity was determined by electrophoresis and observed under UV transilluminator.

Figure 2

Antiviral activity of 3D8 scFv against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in a dose-dependent manner and a cytotoxicity assay. (A) Dose-dependent inhibition of SARS-CoV-2 by 3D8. Vero E6 cells were infected with SARS-CoV-2 at 2 hpi and treated with a range of 3D8 concentrations for 48 hrs. The cells were harvested, and the viral RNA load was determined using RT-qPCR. (B) Supernatants from the 3D8-treated samples were collected, and a plaque assay was performed to determine the infectious viral titer. (C) Percent inhibition of SARS-CoV-2 replication was shown by 3D8 in Vero E6 cells. Replication was measured via quantification of the viral RNA level. (D) Cytotoxicity testing of 3D8 in Vero E6 cells was performed by applying a range of various concentrations in uninfected cell cultures. Data presented here are representative of at least three independent experiments performed in triplicate. Error bars indicate the standard deviation of measurements in the representative experiment. *** p < 0.001, one-way ANOVA test; ns: non-significant.

Figure 3

Prophylactic and therapeutic antiviral effects of 3D8 against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). (A) Inhibition of SARS-CoV-2 by 3D8 in pretreated cell cultures. Vero E6 cells pretreated with 3D8 were infected with SARS-CoV-2. At 48 hpi, the cells were harvested, and the viral copy number was quantified based on the relative concentration of the N gene. (B) The N protein of SARS-CoV-2 was identified by Western blot. (C) Supernatants were harvested from 3D8-pretreated cell cultures infected with SARS-CoV-2, and the infectious viral titer was determined using a plaque assay. The clear zone indicates the plaque formation. (D) Inhibition of SARS-CoV-2 by 3D8 in post-treated cell cultures. Vero E6 cells were infected with SARS-CoV-2 at 2 hpi and treated with 3D8 for 48 hrs. The cells were harvested, and the viral copy number was quantified based on the relative concentration of the N gene. (E) The N protein of SARS-CoV-2 was identified by Western blot. (F) Supernatants were harvested from 3D8-post-treated cell cultures infected with SARS-CoV-2, and the infectious viral titer was determined using a plaque assay. Data presented here are representative of at least three independent experiments performed in triplicate. *** p < 0.001, one-way ANOVA test; ns: non-significant.

Figure 4

In vitro antiviral effect of 3D8 against human coronavirus OC43 (HCoV-OC43). (A) Dose-dependent inhibition of HCoV-OC43 by 3D8. Vero E6 cells were infected with HCoV-OC43 at 2 hpi and treated with a range of 3D8 concentrations for 48 hrs. The cells were harvested, and the virus copy number was determined using qPCR. (B) The N protein of HCoV-OC43 was identified by Western blot. (C) Percent inhibition of HCoV-OC43 replication was shown by 3D8 in Vero E6 cells. Replication was measured via quantification of the viral RNA level. (D) At 48 hpi, the cells were washed with PBS and fixed using methanol. Then, they were permeabilized with buffer and blocked with BSA. The cells were then incubated with primary antibodies overnight. After incubation, TRITC-conjugated anti-mouse and Alexa 488-conjugated anti-rabbit antibodies were added. Hoechst was used to stain the nucleus. Data presented here are representative of at least three independent experiments performed in triplicate. *** p < 0.001; one-way ANOVA test; ns: non-significant.

Figure 5

In vitro antiviral effect of different concentrations of 3D8 against porcine epidemic diarrhea virus (PEDV). (A) Dose-dependent inhibition of PEDV by 3D8. Vero E6 cells were infected with PEDV at 2 hpi and treated with a range of 3D8 concentrations for 48 hrs. The cells were harvested, and the viral RNA load was determined using qPCR. (B) The N protein of PEDV was identified by Western blot. (C) Percent inhibition of PEDV replication was shown by 3D8 in Vero E6 cells. Replication was measured via quantification of the viral RNA level. (D) At 48 hpi, the cells were washed with PBS and fixed with methanol. Then, they were permeabilized with buffer and blocked with BSA. The cells were incubated with primary antibodies overnight. After incubation, TRITC-conjugated anti-mouse and Alexa 488-conjugated anti-rabbit antibodies were added. Hoechst was used to stain the nucleus. Data presented here are representative of at least three independent experiments performed in triplicate. *** p < 0.001; one-way ANOVA test; ns: non-significant.

Figure 6

Suggested mode of action for 3D8. First, 3D8 scFv is internalized into the cell through caveolae-mediated endocytosis. After release from the endosomal compartment, 3D8 binds to the viral nucleic acid and degrades it to prevent its amplification, thus inhibiting viral growth. In addition, 3D8 exerts nuclease activity without sequence specificity and hydrolyzes viral RNA genomes or transcripts.