Antiviral activity of selected antimicrobial peptides against vaccinia virus

Abstract

Antimicrobial peptides (AMPs) are gaining importance as effective therapeutic alternatives to conventional antibiotics. Recently we have shown that a set of nine synthetic antimicrobial peptides, four originating from thrombin-induced human platelet-derived antimicrobial proteins named PD1-PD4 and five synthetic repeats of arginine-tryptophan (RW) repeats (RW1-5) demonstrate antibacterial activity in plasma and platelets. Using WR strain of vaccinia virus (VV) as a model virus for enveloped virus in the present study, we tested the same nine synthetic peptides for their antiviral activity. A cell culture-based standard plaque reduction assay was utilized to estimate antiviral effectiveness of the peptides. Our analysis revealed that peptides PD3, PD4, and RW3 were virucidal against VV with PD3 demonstrating the highest antiviral activity of 100-fold reduction in viral titers, whereas, PD4 and RW3 peptide treatments resulted in 10-30-fold reduction. The EC(50) values of PD3, PD4 and RW3 were found to be 40 microg/ml, 50 microg/ml and 6.5 microM, respectively. In VV-spiked plasma samples, the virucidal activity of PD3, PD4 and RW3 was close to 100% (90-100-fold reduction). Overall, the present study constitutes a new proof-of-concept in developing peptide therapeutics for vaccinia virus infections in biothreat scenarios and as in vitro viral reduction agents.

Fig. 1

Antiviral activity of PD and RW peptides on VV during the pre-infection stage. WR strain of VV was incubated with PD and RW peptides at 100 μg/ml and 10 μM concentration, respectively, for 2 h at RT and titers were measured by counting plaques on B-SC-1 cells. Since PD peptides were of uniform length (15 aa) they were constituted in μg/ml whereas RW peptides though were of varying length (2, 4, 6, 8 and 10 aa) and hence expressed in μM concentrations. These two different concentration expression units were maintained to be consistent with our previously published work (Mohan et al., 2009a). VV infection without the peptides was included as control. PD3, PD4 and RW3 peptides demonstrate significant (p < 0.05, indicated by *) reduction in viral titers.

Fig. 2

Antiviral activity of PD and RW peptides during pre-infection, VV-binding and post-infection stage. PD and RW peptides were added to B-SC-1 cells at 3 different stages: (a) prior to infection (filled bars), (b) during virus binding (open bars) or (c) post-infection stage (shaded bars) and resulting viral titers were compared to that of VV-infection lacking the peptides. In the pre-infection experiment B-SC-1 cells were incubated with PD and RW peptides for 2 h at 37 °C, followed by washing of the cells and infection with VV. For the virus-binding stage experiment peptides were mixed individually with VV and the mixture was added to cells. Following incubation for 1 h inoculum was aspirated and agar overlay with DMEM was added. The post-infection assay was performed by first infecting B-SC-1 cells with VV for 1 h and then adding fresh medium containing PD and RW peptides. Note that both PD and RW peptides do not exhibit significant antiviral activity (p > 0.05) during these three stages of treatment.

Fig. 3

Dose–response curves or EC₅₀ estimation of PD and RW peptides. Serial doubling dilution analysis was performed with PD3, PD4 and RW3 peptides. The final concentration of the PD peptides for the assay were 100, 50, 25 and 12.5 μg/ml whereas the RW peptides concentration was 10, 5, 2.5 and 1.25 μM. 1 ml of WR virus (10³ pfu/ml) was mixed individually with each of these peptides at RT for 2 h, followed by infection of B-SC-1 cells for 1 h. Following virus binding agar overlay with DMEM was added to cells and incubated at 37 °C for 72 h. Viral titers were estimated by counting plaques at the four different concentrations of the peptides tested and EC₅₀ values were deduced by using the GraphPad Prism 5.0 software. Peptide concentrations are represented on the x-axis (log scale) with PD3 and PD4 expressed in μg/ml and RW3 concentration expressed in μM. Analysis reveals that the EC₅₀ values for PD3 (A), PD4 (B) and RW3 (C) were 40 μg/ml, 50 μg/ml and 6.5 μM, respectively.

Fig. 4

Antiviral activity of PD and RW peptides against VV in spiked-plasma samples. Human plasma samples spiked with 10² pfu of VV-WR virus were incubated with PD3, PD4 and RW3 peptides individually for 2 h and tested for antiviral activity by infecting B-SC-1 cells and measuring virus titer by performing the plaque assay as described in the pre-infection experiment. Note that PD3, PD4 and RW3 treatment results in 90–100-fold reduction of viral titers (p < 0.05, indicated by *).