Because VSV-Δ51 has been documented as a potent oncolytic virus with a short replication lifecycle, we wanted to determine whether the incorporation of the SARS-CoV-2 RBD gene into the VSV-Δ51 viral genome would improve the viral spreading capacity. After we confirmed the expression of the RBD protein by Western blot (our unpublished data), a conventional viral plaque assay of VSV-Δ51 or VSV-Δ51-RBD viruses in VERO cells was carried out, and the size of the viral plaques were measured using ViralPlaque. Surprisingly, the surface area of VSV-Δ51-RBD plaques was significantly larger than that of the plaques formed by the VSV-Δ51 virus, with a mean surface area of 1.962 mm² vs. 1.279 mm², respectively ( Figure 1 ). This observation suggests that the SARS-CoV-2 RBD favors viral spreading.

To determine whether the enhanced viral spreading would be translated to an increase in viral yield, we evaluated the total virus yield from VERO cells infected with either VSV-Δ51 or VSV-Δ51-RBD viruses. We measured the virus yield at different tissue culture scales ranging from 96 wells, with a surface area of 0.32 cm², to 10 layers of cellSTACKs, with a surface area of 6,350 cm². As expected, VSV-Δ51-RBD-infected VERO cells had significantly higher virus titers than VSV-Δ51-infected VERO cells across all of the different culture scales as follows: 96-well format, 12-well format, 6-well format, 15-cm plates and 10 layers of cellSTACKs (P < 0.05, *P < 0.05, **P < 0.005, ****P < 0.00005, respectively) ( Figure 2 ). This finding suggests that the SARS-CoV-2 RBD could be utilized to improve viral yields for large-scale virus production.

To determine the therapeutic potential of the SARS-CoV-2 RBD, we evaluated the cytotoxicity of the VSV-Δ51-RBD and VSV-Δ51 viruses using three different cancer cell lines (LLC1 – a murine lung cancer cell line, A549 – a human lung cancer cell line and B16F10 – a murine melanoma cell line). Based on data from the CellTiter-Glo^® Luminescent cell viability assay, we demonstrated that the in vitro cytotoxicity of the VSV-Δ51-RBD virus was significantly higher (*P < 0.05) than that of the VSV-Δ51 virus at low MOIs (0.001, 0.01 and 0.1) ( Figure 3 ). To further validate the contribution of the SARS-CoV-2 RBD to this phenomenon, we examined the cytotoxicity of the VSV-Δ51 virus in the VERO cell line in the presence of exogenous recombinant RBD protein. In alignment with the cytotoxicity results for the VSV-Δ51-RBD virus shown in ( Figure 3 ), the addition of a recombinant RBD protein at doses of 120 ng/well, 80 ng/well and 40 ng/well significantly enhanced the cytotoxicity of VSV-Δ51 virus in a dose-dependent manner compared to infection with VSV-Δ51 alone but to a limited level compared to VSV-Δ51-RBD (*P < 0.05, ***P < 0.0005, ****P < 0.00005, respectively) ( Figure 3 ). This finding is supported by the observation that the addition of anti-RBD neutralizing antibody to the culture at different dilutions abrogated the effect of the recombinant RBD protein only at a high concentration; 12.5ng/well (*P < 0.005). Interestingly, the addition of anti-RBD neutralizing antibody to the VSV-Δ51-RBD, even at a high concentration, did not completely eliminate the cytotoxicity of the VSV-Δ51-RBD to the same level as the VSV-Δ51 virus, suggesting that the RBD-enhanced toxicity is more pronounced when the RBD protein is expressed within the cytoplasm rather than when supplemented extracellularly. We also found that the recombinant RBD alone has limited cytotoxicity compared to uninfected cells (~ 93%). This suggests that RBD alone does not have strong cytotoxic activity, and its enhancement when expressed within the VSV genome is likely due to its ability to enhance viral replication and spread rather than direct cytotoxicity. These findings collectively support the potential utility of SARS-CoV-2 RBD, incorporated into the viral genome, for enhancing viral cytotoxicity.

To evaluate the oncolytic activity of VSV-Δ51-RBD in vivo, B16F10 cells infected with either VSV-Δ51 or VSV-Δ51-RBD. These virus-infected cells were injected into the peritoneal cavity of C57BL/6 mice. Mice injected with VSV-Δ51-RBD-infected B16F10 cells had a significantly improved survival benefit compared to mice injected with VSV-Δ51-infected B16F10 cells (**P < 0.01) ( Figure 4A ). To further test the efficacy of VSV-Δ51-RBD in a clinically relevant tumor model, we established subcutaneous B16F10 tumors and mice were then subsequently treated with a single dose of either VSV-Δ51 or VSV-Δ51-RBD intratumorally 10 days after tumor implantation. Overall, mice treated with either VSV-Δ51 or VSV-Δ51-RBD had a significantly improved survival outcome compared to those receiving no treatment, with a median survival of 31 days and 38 days post-tumor seeding, respectively ( Figure 4B ). Even though there was a statistically significant difference between the VSV-Δ51 and VSV-Δ51-RBD treatments (*P < 0.05), there was no complete tumor remission in the VSV-Δ51-RBD-treated group. Altogether, these observations further support the therapeutic potential of the SARS-CoV-2 RBD.

A549 human lung carcinoma and VERO cell lines were generous gifts from Mr. Suhail Melibary (King Abdulaziz University Hospital, Jeddah, Saudi Arabia). The LLC1 and B16F10 murine cell lines were generous gifts from Dr. Nada Zaidan and Dr. Khalid Shah (KACST-BWH Centre of Excellence for Biomedicine, Riyadh, Saudi Arabia). All cell lines were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Sigma Aldrich, Saudi Arabia).

VERO cells were seeded into 6-well plates at a density of 5 × 10⁵ cells per well. The next day, cell monolayers were infected with 500 μl of serially diluted viruses in serum-free DMEM for 1 hour. After viral adsorption by the cells, the infectants were removed, and 2 ml of 1% agarose overlay was added (2X Dulbecco’s modified Eagle’s medium and 20% FBS: 2% agarose at a 1:1 ratio). At 48 hours post-infection (hpi), the cells were fixed with 4% formaldehyde and stained with crystal violet. Plates were washed with tap water, dried and scanned using a flatbed office scanner at a resolution of 300 dots per inch (dpi). Plaques were counted, and the surface area was measured using ViralPlaque (LowRes method, single-well mode and manual control option on). All false positive plaques were eliminated manually. Finally, the area (in pixels squared) was transformed into diameter units (mm) assuming circular plaques.

The VSV-Δ51-RBD virus was created in Alkayyal’s and Mahmoud’s labs by inserting a codon-optimized RBD gene, containing the amino acid region (319-541aa) of the full Spike gene of the SARS-CoV-2 (2019-nCoV), from the RBD expression plasmid (Sino Biological Inc., Beijing, China, cat# VG40592-UT) into a plasmid encoding the VSV-Δ51 antigenome plasmid, which was a gift from Dr. Anwar Hashim (King Abdulaziz University, Jeddah, Saudi Arabia). The insertion was between the G and L genes, and primers used for this insertion as follows: Forward 5’-TGGAAAGTAAGCTAGCTGTATGAAAAAAACTCATCAACAGCCATCATGAGGGTCCAACCA-3’ and Reverse: 5’-GAAGAATCTGGCTAGCTCAGAAGTTCACACACTTGTTC-3’. These primers were designed to be compatible with the In-Fusion^® HD Cloning Kit (Takara Bio Inc, USA, cat# 638910). Both viruses were rescued in Alkayyal’s and Mahmoud’s labs as described elsewhere (16), and were grown in VERO cells by infecting cells at 0.1 MOI and subsequently purified from the supernatant by a sucrose gradient. Virus pellets were resuspended in PBS, aliquoted and kept at –80°C until used.

The A549, LLC1, B16F10 or VERO cell lines were seeded into 96-well plates at a density of 2 × 10⁴ cells/well. The next day, cells were infected with either VSV-Δ51 or VSV-Δ51-RBD at various MOIs ranging from 0.0001–100 pfu/cell. For the assessment of the recombinant RBD effects on the VSV-Δ51 cytotoxicity, VERO cells pretreated with ascending concentrations of the recombinant RBD protein (Sino Biological Inc., Beijing, China, cat# 40592-VNAH) as indicated in ( Figure 3D ) and infected with VSV-Δ51 at 0.01 MOI. When needed, the neutralizing anti-RBD antibodies (Acrobiosystems Inc, Newark, USA, cat# SPD-M128) were preincubated with VSV-Δ51-RBD or the recombinant RBD protein for one hour at 37°C, then added to VERO cells. Following a 48-hour incubation, CellTiter-Glo^® Luminescent Cell Viability Assay reagent (Promega, cat# G7571) was used to quantify the amount of ATP present in the cultures, which is reflective of the number of metabolically active cells. Briefly, 100 µl of CellTiter-Glo^® reagent was added to each well and incubated at room temperature for 12 minutes, two minutes of which were on an orbital shaker to induce cell lysis.

B16F10 tumor cells were harvested from tissue culture plates, resuspended in PBS at a concentration of 1 × 10⁸ cells/ml, and 200 μl was aliquoted into Eppendorf tubes. Cells were then infected with VSV-Δ51 or VSV-Δ51-RBD at an MOI of 10 followed by incubation for 2 hours at 37°C. Next, 100 μl of the infected cells was injected into mice intraperitoneally. Therefore, each dose contained 5 × 10⁶ B16F10 cells infected with 5 × 10⁷ pfu of the virus. The survival of the mice was monitored, and the endpoint was defined as when peritoneal distension developed. For the subcutaneous tumor model, tumors were established by injecting 5 × 10⁵ B16F10 cells in 100 µl PBS subcutaneously. Treatment began on day 10 after tumor implantation, with a single injection of VSV-Δ51 or VSV-Δ51-RBD viruses at a concentration of 1 × 10⁸ pfu.

All statistical analyses were performed using GraphPad Prism 9.0 software. The Student’s t-test was used to determine statistical significance, with a cutoff of P = 0.05. Data are presented as ± SD. For the survival analysis, Kaplan-Meier survival curves were used and statistical analysis was performed using the Log-rank (Mantel-Cox) test, with a cutoff of P = 0.05.