Development of novel SARS-CoV-2 viral vectors

Abstract

The authentic SARS-CoV-2 requires to be handled in Biosafety Level 3 laboratories, which restrains investigation by the broader scientific community. Here, we report the development of a novel SARS-CoV-2 viral vector composed of all 4 SARS-CoV-2 structural proteins, the packaging signal sequence of SARS-CoV-2, a reporter gene, and an RNA amplification component of Venezuelan equine encephalitis virus (VEEV). This VEE-SARS-CoV-2 viral vector transduces target cells in an ACE2-dependent manner, and all 4 structural proteins of SARS-CoV-2 are indispensable for its transduction activity. Comparative studies show that the incorporation of the VEEV self-amplification mechanism increases the gene expression level by ~ 65-fold and extends the transgene expression up to 11 days in transduced cells. Additionally, we demonstrated the significant applications of this new VEE-SARS-CoV-2 vector for neutralizing antibody quantification and antiviral drug testing. The VEE-SARS-CoV-2 vectors developed will be an important and versatile tool for investigating SARS-CoV-2 molecular virology, developing antiviral agents targeting receptor binding, and studying RNA genome packaging and function of the essential but not well studied structural proteins of SARS-CoV-2.

Figure 1

Design of VEE-SARS-CoV-2 viral vectors. (A) Schematic diagram of the core elements in VEE-SARS-CoV-2 plasmid including a CMV promoter, 5’ UTR and nsP1-4 gene of VEEV, 26 subgenomic VEEV promoter (26S), reporter genes (Luc or GFP), the SARS-CoV-2 packaging signal (PS9 sequence), 3’ UTR of VEEV genome, and a poly(A) signal. (B) Mechanistic illustration of production of VEE-SARS-CoV-2 viral vectors. Components of viral vectors were produced in 293T cells by co-transfection with plasmids carrying structural protein genes (S, M-E, N) and reporter gene with regulatory elements for amplification and expression. The viral vectors are assembled and released from 293T cells. (C) Schematic presentation of transduction of viral vectors. RNA amplification and reporter gene expression are accomplished in target cells via ACE2-mediated internalization, translation of VEEV nsP1-4 genes, formation of VEEV nsP1-4 protein complex, and synthesis of the negative-strand RNA. The newly synthesized negative-strand RNA serves as the template for synthesis of positive strand RNA and production of sub-genomic RNA encoding reporter gene.

Figure 2

Optimization of VEE-SARS-CoV-2 viral vector production. (A) Optimization of the ratio between the plasmid carrying spike protein gene and the rest of plasmids including those carrying M-E, N, and the transfer plasmid for transduction activity. (B) Viral vector titer. (C) Luciferase activity per 10⁶ genome copies of viral vectors. (D) Optimization of transduction activity. (E) Titer of viral vectors at different mass ratios of each plasmid with fixed amount of spike plasmid. (F) Luciferase activity per 10⁶ genome copies of vectors. Data are presented as the mean ± SD of 3 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey Post Hoc tests. **p < 0.01, ***p < 0.001.

Figure 3

Characterization of VEE-SARS-CoV-2 viral vectors. (A–C) The effects of structural gene and package signal sequence on transduction activity of VEE-SARS-CoV-2 vectors prepared by co-transfection of 293T cells with all but lacking one structural gene or package signal sequence. The viral vectors were prepared and titrated by qPCR and examined for transduction activity in ACE2-TMPRSS2-293T cells. (D) Cell type-dependent transduction activity of VEE-SARS-CoV-2 viral vector. Same number of cells (2 × 10⁵ /well) were transduced with equal number of VEE-SARS-CoV-2-Luc vectors. The transduction activity was determined by luciferase assay at 48 h post-transduction. (E) Dose-dependent transduction of VEE-SARS-CoV-2 viral vectors. Same number of ACE2-TMPRSS2-293T cells (2 × 10⁵ /well) were transduced with increasing amount of VEE-SARS-CoV-2-Luc vectors and luciferase activity was determined. (F) Cytotoxicity assessment of VEE-SARS-CoV-2-Luc vectors in ACE2-TMPRSS2-293T cells. Lactate dehydrogenase (LDH) activity in the supernatant of transduced cells was determined 48 h post-transduction. Cell lysate was used as the positive control for total release of LDH. (G) Luciferase gene expression as function of MOI. (H) VEE-SARS-CoV-2-Luc vector stability under different conditions. All luciferase activity in transduced cells was measured 24 h post-transduction except for (D) and (F). Data are presented as the mean ± SD of 3 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey Post Hoc tests. ***p < 0.001.

Figure 4

Comparison of time-dependent reporter gene expression in ACE2-TMPRSS2-293T cells transduced by VEE-SARS-CoV-2 vectors and SC2-VLPs. (A) Luciferase gene expression level and (B) folds of luciferase mRNA change in ACE2-TMPRSS2-293T cells after transduction by VEE-SARS-COV-2-Luc vectors and SC2-Luc-VLP. Transduced cells were harvested at the indicated times, and luciferase assay and real-time qPCR analysis were performed. The fold change was normalized to the first time point determined (2 h post-transduction). GAPDH was used as the internal control. Data are presented as the mean ± SD of independent triplicates. (C) GFP expression in ACE2-TMPRSS2-293T cells transduced with VEE-SARS-CoV-2-GFP vectors or SC2-GFP VLPs at 48 h post-transduction. (D) Time-dependent GFP expression in ACE2-TMPRSS2-293T cells transduced by VEE-SARS-CoV-2-GFP vectors. Transduced cells were passaged every 4 days and GFP expression was recorded under a fluorescence microscope and cell density was photographed under regular microscope at the indicated times. Scale bar = 200 μm.

Figure 5

Inhibition of VEE-SARS-CoV-2-Luc-mediated transduction by neutralizing antibodies (A) and antiviral agents (B). VEE-SARS-CoV-2-Luc vectors were incubated with MM43 SARS-CoV-2 spike neutralizing antibody, antiviral compound of cepharanthine, fangchiboline, or berbamine hydrochloride, for 1 h in a volume of 100 µL at indicated concentration. The mixture was added to ACE2-TMPRSS2-293T cells (2.5 × 10⁴ /well) pre-seeded one day earlier. Transduction proceeded for 3 h, and the medium was replaced with fresh medium. Luciferase activity was determined at 48 h post-transduction. (C) Cytotoxicity of compounds at different concentrations was determined at 48 h post-treatment by LDH cytotoxicity assay. Data are presented as the mean ± SD of independent triplicates.